Cross-beta structure comprising amyloid-binding proteins and methods for detection of the cross-beta structure, for modulating cross-beta structures fibril formation and for modulating cross-beta structure-mediated toxicity

ABSTRACT

The invention relates to the field of biochemistry, molecular biology, structural biology and medicine. More in particular, the invention relates to cross-β structures and the biological role of these cross-β structures. In one embodiment, the invention discloses a method for modulating extracellular protein degradation and/or protein clearance comprising modulating cross-β(beta) structure formation (and/or cross-β structure-mediated activity) of the protein present in the circulation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT International PatentApplication PCT/NL2003/000501, filed Jul. 8, 2003, designating theUnited States of America, corresponding to PCT International PublicationWO 2004/004698 A3 (published in English on Jan. 15, 2004), the contentsof the entirety of which are incorporated by this reference.

TECHNICAL FIELD

The invention relates to the fields of biotechnology, biochemistry,molecular biology, structural biology and medicine. More in particular,the invention relates to cross-β structure, their binding proteins andtheir biological roles.

BACKGROUND

An increasing body of evidence suggests that unfolding of globularproteins can lead to toxicity.¹ Unfolded proteins can initiate proteinaggregation and fibrillization by adopting a partially structuredconformation. Such fibrillar aggregates can (slowly) accumulate invarious tissue types and are associated with a variety of degenerativediseases. The term “amyloid” is used to describe these fibrillardeposits (or plaques). Diseases characterized by amyloids are referredto as amyloidosis and include Alzheimer disease (AD), light-chainamyloidosis, type II diabetes and spongiform encephalopathies. It hasbeen found recently that toxicity is an inherent property of misfoldedproteins. According to the present invention, this is the commonmechanism for these conformational diseases.¹

A cross-β structure is a secondary structural element in peptides orproteins. A cross-β structure can be formed upon denaturation,proteolysis or unfolding of proteins.² These secondary structureelements are typically absent in globular regions of proteins. Thecross-β structure is found in amyloid fibrils. Amyloid peptides orproteins are cytotoxic to cells. A cross-β structure includes stackedβ-sheets. In a cross-β structure, the individual β-strands either runperpendicular to the long axis of a fibril or run in parallel to thelong axis of a fiber. The direction of the stacking of the β-sheets incross-β structures is perpendicular to the long fiber axis.

SUMMARY OF THE INVENTION

It is reported herein that glycation of proteins also induces theformation of the cross-β structure. These results, combined withexisting literature information, indicate that a common structure isinduced upon unfolding of globular proteins. Therefore, the presentinvention discloses a novel pathway involving a cross-β structure, whichpathway will be called a “cross-β structure pathway.” This pathwayincludes several cross-β structure-binding proteins, including so-calledmultiligand receptors, and is involved in protein degradation and/orprotein clearance. Also reported herein is the identification of novelcross-β-binding proteins that contain a cross-β structure-bindingmodule. These findings support the identification of a cross-β structurepathway. Multiple aspects of this novel pathway are outlined below.

In one embodiment, the present invention discloses that proteolyzed,denatured, unfolded, glycated, oxidized, acetylated or otherwisestructurally altered proteins adopt cross-β structures. Examples ofknown cross-β structure-forming proteins are all proteins that causeamyloidosis or proteins that are found in disease-related amyloiddepositions, for example, but not restricted to, Alzheimer β-amyloid(Aβ) and Islet Amyloid PolyPeptide (IAPP). The present inventiondiscloses that fibrin, glycated proteins (for example, glycated albuminand glycated hemoglobin), and endostatin are also capable of adopting across-β structure.

The invention furthermore discloses the identification of the formationof a cross-β structure as a signal for protein degradation and/orprotein clearance.

The serine protease tissue plasminogen activator (tPA) induces theformation of plasmin through cleavage of plasminogen. Plasmin cleavesfibrin and this occurs during lysis of a blood clot. Although notessential for fibrinolysis in mice,^(3,4) tPA has been recognized forits role in fibrinolysis for a long time.^(5,6) Activation ofplasminogen by tPA is stimulated by fibrin or fibrin fragments, but notby its precursor, fibrinogen.⁷⁻¹⁰ This can be in part explained by thestrong binding of tPA to fibrin and weak binding to fibrinogen. Thebinding sites in fibrin and in tPA responsible for binding andactivation of tPA have been mapped and studied in detail.⁸⁻²¹ However,the exact structural basis for the interaction of tPA with fibrin wasunknown. In addition to fibrin and fibrin fragments, many other proteinshave been described that are similarly capable of binding tPA andstimulating tPA-mediated plasmin formation.²²⁻³⁶ Like with fibrin andfibrin fragments, the exact nature of the interaction(s) between theseligands for tPA and tPA were not known. Moreover, it was unknown why andhow all of these proteins, which lack primary sequence homology, bindtPA. The present invention discloses tissue-type plasminogen activator(tPA) as a protein capable of binding cross-β structures. Furthermore,the invention discloses the finger domain (also named fibronectin type Idomain) and other comparable finger domains as a cross-βstructure-binding module. The present invention further discloses thatproteins which bind to these fingers will be typically capable offorming cross-β structures.

Since fibrin contains the cross-β structure, the present invention alsodiscloses that the generation of cross-β structures plays a role inphysiological processes. The invention discloses that the generation ofcross-β structures is part of a signaling pathway, the “cross-βstructure pathway,” that regulates protein degradation and/or proteinclearance. Inadequate function of this pathway may result in thedevelopment of diseases, such as conformational diseases³⁷ and/oramyloidosis.

The present invention furthermore discloses that the cross-β structureis a common denominator in ligands for multiligand receptors.³⁸ Theinvention discloses, therefore, that multiligand receptors belong to the“cross-β structure pathway.”

The best studied example of a receptor for a cross-β structure isRAGE.³⁹⁻⁴⁴ Examples of ligands for RAGE are Aβ, protein-advancedglycation end-products (AGE) adducts (including glycated-BSA),amphoterin and S100. RAGE is a member of a larger family of multiligandreceptors³⁸ that includes several other receptors, some of which,including CD36, are known to bind cross-β structure-containing proteins(see also FIG. 1). At present, it is not clear what the exact nature ofthe structure or structures is in the ligands of these receptors thatmediates the binding to these receptors. It is reported herein thatglycation of proteins also induces the formation of a cross-β structure.Therefore, it is disclosed that all of these receptors form part of amechanism to deal with the destruction and removal of unwanted or evendamaging proteins or agents. These receptors play a role in recognitionof infectious agents or cells, recognition of apoptotic cells and ininternalization of protein complexes and/or pathogens. It is furthermoredisclosed that all of these receptors recognize the same or similarstructure, the cross-β structure, to respond to undesired molecules. Itis shown herein that tPA binds cross-β structures, providing evidencethat tPA belongs to the multiligand receptor family. As disclosedherein, tPA and the other multiligand receptors bind the cross-βstructure and participate in the destruction of unwanted biomolecules. Aprominent role of the protease tPA in the pathway lies in its ability toinitiate a proteolytic cascade that includes the formation of plasmin.Proteolysis is likely to be essential for the degradation and subsequentremoval of extracellular matrix components. The effect of tPA on theextracellular matrix will affect cell adhesion, cell migration, cellsurvival and cell death, through, for example, integrin-mediatedprocesses. Based on these studies, strong evidence is provided that atleast three other proteins, FXII (factor XII), hepatocyte growth factoractivator (HGFa), and fibronectin, that contain one or more fingerdomain(s) are also part of the “cross-β structure pathway.”

The role of FXII is especially important, since it activates theintrinsic coagulation pathway. Activation of the intrinsic pathway, theresulting formation of vasoactive peptides, and the activation of otherimportant proteins contribute to the process of protection and/orclearance of undesired proteins or agents. The “cross-β structurepathway” is modulated in many ways. Factors that regulate the pathwayinclude modulators of synthesis and secretion, as well as modulators ofactivity. The pathway is involved in many physiological and pathologicalprocesses. Therefore, the invention furthermore provides a method formodulating extracellular protein degradation and/or protein clearancecomprising modulating the activity of a receptor for cross-βstructure-forming proteins. Examples of receptors for cross-βstructure-forming proteins include RAGE, CD36, Low densitylipoprotein-Related Protein (LRP), Scavenger Receptor B-1 (SR-B1), andSR-A. The invention discloses that FXII, HGFa and fibronectin are alsoreceptors for cross-β structures.

The present invention discloses that tissue-type plasminogen activator(tPA) is a cross-β structure-binding protein, a multiligand receptor anda member of the “cross-β structure pathway.” The invention disclosesthat tPA mediates cross-β structure-induced cell dysfunction and/or celltoxicity. The invention discloses that tPA mediates, at least in part,cell dysfunction and/or toxicity through activation of plasminogen. Theplasminogen-dependent effects are inhibited by B-type carboxypeptidaseactivity B and, thus, a role for carboxyterminal lysine residues in thecross-β structure pathway is disclosed.

The present invention relates, amongst others, to the structure(s) infibrin and other proteins that bind tPA, to the binding domain in tPA,and to the pathway(s) regulated by this structure. The present inventiondiscloses a presence of cross-β structures in proteins and peptides thatare capable of binding tPA. The herein-disclosed results indicate astrong correlation between the presence of a cross-β structure and theability of a molecule to bind tPA. Furthermore, the results indicate thepresence of an amyloid structure in fibrin. This indicates that underphysiological conditions, a cross-β structure can form, a phenomenonthat has been previously unrecognized. The formation of cross-βstructures has thus far only been associated with severe pathologicaldisorders. tPA binds denatured proteins, which indicates that a largenumber of proteins, if not all proteins, can adopt a conformationcontaining cross-β structures or cross-β-like structure(s). Takentogether, the formation of cross-β structures is likely to initiateand/or participate in a physiological cascade of events necessary toadequately deal with removal of unwanted molecules, i.e., misfoldedproteins, apoptotic cells or even pathogens. FIG. 1 shows a schematicrepresentation of the “cross-β structure pathway.” This pathwayregulates the removal of unwanted biomolecules during several processes,including fibrinolysis, formation of neuronal synaptic networks,clearance of used, unwanted and/or destroyed (denatured) proteins,induction of apoptosis and clearance of apoptotic cells and pathogens.If insufficiently or incorrectly regulated or disbalanced, the pathwaymay lead to severe disease.

Thus, in a first embodiment, the invention discloses a method formodulating extracellular protein degradation and/or protein clearancecomprising modulating cross-β(beta) structure formation (and/or cross-βstructure-mediated activity) of the protein present in the circulation.

There are two major regular protein-folding patterns, which are known asthe β(beta)-sheet and the a-helix. An antiparallel β-sheet is formedwhen an extended polypeptide chain folds back and forth upon itself;with each section of the chains running in the direction opposite tothat of its immediate neighbors. This gives a structure held together byhydrogen bonds that connect the peptide bonds in neighboring chains.Regions of a polypeptide chain that run in the same direction form aparallel β-sheet. A cross-β structure is composed of stacked β-sheets.In a cross-β structure, the individual β-strands either runperpendicular to the long axis of a fibril or run in parallel to thelong axis of a fiber. The direction of the stacking of the β-sheets incross-β structures is perpendicular to the long fiber axis. As disclosedherein, a broad range of proteins is capable of adopting a cross-βstructure and, moreover, these cross-β structure-comprising proteins areall capable of binding and stimulating tPA, thus promoting destructionof unwanted or damaging proteins or agents.

An extracellular protein is typically defined as a protein presentoutside a cell or cells.

Protein degradation and/or protein clearance includes the breakdown andremoval of unwanted proteins, for example, unwanted and/or destroyed(for example, denatured) proteins. Also included is the removal ofunwanted biomolecules during several processes, including fibrinolysis,formation of neuronal synaptic networks, clearance of used, unwantedand/or destroyed (denatured) proteins, induction of apoptosis andclearance of apoptotic cells and pathogens.

The term “in the circulation” is herein defined as a circulation outsidea cell or cells, for example, but not restricted to, the continuousmovement of blood.

In yet another embodiment, the invention discloses a method forincreasing extracellular protein degradation and/or protein clearancecomprising increasing cross-β structure formation and/or cross-βstructure-mediated activity of the protein present in the circulation.Increase of cross-β structure formation of a particular protein leads,for example, to activation of tPA, which, in turn, induces the formationof plasmin through cleavage of plasminogen and thus results in anincrease in the degradation and/or protein clearance.

In one embodiment, the invention discloses a method for increasingextracellular protein degradation and/or protein clearance comprisingproviding a compound capable of increasing cross-β structure formation(and/or cross-β structure-mediated activity) of the protein present inthe circulation. In another embodiment, the compound capable ofincreasing cross-β structure formation is glucose. Under certaincircumstances, the addition of glucose to a protein leads to anirreversible, non-enzymatic glycation reaction in which predominantly aglucose molecule is attached to the free amino groups of lysine residuesin a protein. In addition, N-termini and free amino groups of arginineresidues are prone to glycation. It is disclosed herein within theexperimental part that glycation leads to cross-β structure formation.Hence, the invention discloses a method for increasing extracellularprotein degradation and/or protein clearance comprising providing acompound capable of increasing cross-β structure formation of theprotein present in the circulation.

Other examples of compounds capable of increasing (or mimicking) cross-βstructure formation in a protein are apolar solutions, urea (asdisclosed herein within the experimental part), and ions (for exampleZn²⁺). However, it is clear that there are also other ways to increaseor mimic cross-β structure formation, for example, by denaturation, lowpH, temperature, mutations or protein modification in general (forexample, oxidation).

In addition to a method for increasing extracellular protein degradationand/or protein clearance comprising increasing cross-β structureformation of the protein present in the circulation via any of theabove-described methods to degrade and/or remove, preferably, theprotein which comprises the cross-β structure, it is also possible todegrade and/or remove a protein which does not comprise a cross-βstructure. This is, for example, accomplished by providing a compoundcomprising a cross-β structure and a compound comprising tPA-likeactivity at or near the protein which needs to be degraded and/orremoved. An example of a compound comprising a cross-β structure isfibrin or a fragment thereof comprising the cross-β structure. Anexample of a compound comprising tPA-like activity is tPA.

In another embodiment, the invention discloses a method for decreasingextracellular protein degradation and/or protein clearance comprisingdecreasing cross-β structure formation of the protein present in thecirculation. For instance, the invention discloses a method fordecreasing extracellular protein degradation and/or protein clearancecomprising providing a compound capable of decreasing cross-β structureformation of the protein present in the circulation. Decreasing ofcross-β structure formation is, for example, accomplished by shieldingor blocking of the groups involved in the formation of a cross-βstructure. Examples of compounds capable of decreasing cross-β structureformation are Congo red, antibodies, β-breakers, phosphonates, heparin,amino-guanidine or laminin.⁴⁵ Yet another way to decrease cross-βstructure formation in a protein is by removal of a glucose groupinvolved in the glycation of the protein.

In yet another embodiment, the invention discloses a method formodulating extracellular protein degradation and/or protein clearancecomprising modulating tPA or tPA-like activity. tPA induces theformation of plasmin through cleavage of plasminogen. Plasmin cleavesfibrin and this occurs during lysis of a blood clot. Activation ofplasminogen by tPA is stimulated by fibrin or fibrin fragments, but notby its precursor fibrinogen. The term “tPA-like activity” is hereindefined as a compound capable of inducing the formation of plasmin,possibly in different amounts, and/or other tPA-mediated activities.Preferably, tPA-like activity is modified such that it has a higheractivity or affinity towards its substrate and/or a cofactor. This is,for example, accomplished by providing the tPA-like activity withmultiple binding domains for cross-β structure-comprising proteins.Preferably, the tPA-like activity is provided with multiple fingerdomains. It is herein disclosed that the three-dimensional structures ofthe tPA finger domain and the fibronectin finger domains 4-5 revealstriking structural homology with respect to local charge-densitydistribution. Both structures contain a similar solvent-exposed stretchof five amino acid residues with alternating charge; for tPA, Arg7,Glu9, Arg23, Glu32, Arg30, and for fibronectin, Arg83, Glu85, Lys87,Glu89, Arg90, located at the fifth finger domain, respectively. Thecharged-residue alignments are located at the same side of the fingermodule. Hence, the tPA-like activity is provided with one or more extrafinger domain(s) which comprise(s) ArgXGlu(X)13Arg(X)8GluXArg (SEQ IDNO: 1) or ArgXGluXLysXGluArg (SEQ ID NO: 2).

The activity of tPA and/or the tPA-mediated activation of plasminogen isincreased by the binding to fibrin fragments or other protein fragmentsthat have a lysine or an arginine at the carboxy-terminal end. B-typecarboxypeptidases, including, but not limited to, carboxypeptidase B(CpB) or Thrombin Activatable Fibrinolysis Inhibitor (TAFI, also namedcarboxypeptidase U or carboxypeptidase R), are enzymes that cleave offcarboxy-terminal lysine and arginine residues of fibrin fragments thatwould otherwise bind to tPA and/or plasminogen and stimulate plasminformation.

In one embodiment, the invention discloses a method for increasingextracellular protein degradation and/or protein clearance comprisingproviding a compound capable of increasing tPA-like and/or tPA-mediatedactivity or activities. In another embodiment, the invention discloses amethod for increasing extracellular protein degradation and/or proteinclearance comprising providing a compound capable of increasing tPA-likeactivity, wherein the compound comprises a cross-β structure. In anotherembodiment, the invention discloses a method for increasingextracellular protein degradation and/or protein clearance comprisingproviding a compound capable of inhibiting B-type carboxypeptidaseactivity. In an additional embodiment, the compound comprisescarboxypeptidase inhibitor (CPI) activity.

In yet another embodiment, the invention discloses a method fordecreasing extracellular protein degradation and/or protein clearancecomprising providing a compound capable of decreasing tPA-like activity.In one aspect, the invention discloses a method for decreasingextracellular protein degradation and/or protein clearance comprisingproviding a compound capable of decreasing tPA-like activity ortPA-mediated activity or activities, wherein the compound is a proteinand/or a functional equivalent and/or a functional fragment thereof. Forexample, such a compound capable of decreasing tPA-like activity is aninhibitor of tPA or a substrate of tPA which binds and does not let go.Examples of a compound capable of decreasing tPA-like activity ortPA-mediated activity include, but are not limited to, lysine, arginine,e-amino-caproic acid or tranexamic acid, serpins (for example,neuroserpin, PAI-1), tPA-Pevabloc, antibodies that inhibit tPA-likeactivity or tPA-mediated activity or B-type carboxypeptidase(s). Forexample, providing lysine results in the prevention or inhibition ofbinding of a protein comprising a C-terminal lysine-residue to theKringle domain of plasminogen. Hence, tPA activation is prevented orinhibited. Preferably, the compound capable of decreasing tPA-likeactivity or tPA-mediated activity or activities reduce the tPA-likeactivity or tPA-mediated activity or activities and, even morepreferably, the tPA-like activity or tPA-mediated activity or activitiesis completely inhibited.

A functional fragment and/or a functional equivalent are typicallydefined as a fragment and/or an equivalent capable of performing thesame function, possibly in different amounts. For example, a functionalfragment of an antibody capable of binding to a cross-β structure wouldbe the Fab′ fragment of the antibody.

In yet another embodiment, the invention discloses a method formodulating extracellular protein degradation and/or protein clearancecomprising modulating an interaction between a compound comprising across-β structure and a compound comprising tPA-like activity. Inanother embodiment, the invention discloses a method for decreasingextracellular protein degradation and/or protein clearance comprisingdecreasing an interaction between a compound comprising a cross-βstructure and a compound comprising tPA-like activity. Such a compoundis, for example, a chemical, a proteinaceous substance or a combinationthereof. In an additional embodiment, the invention discloses a methodfor decreasing extracellular protein degradation and/or proteinclearance comprising providing a compound capable of decreasing aninteraction between a compound comprising a cross-β structure and acompound comprising tPA-like activity. In one aspect, the inventiondiscloses a method for decreasing extracellular protein degradationand/or protein clearance according to the invention, wherein thecompound is a protein and/or a functional equivalent and/or a functionalfragment thereof. In another aspect, the protein is an antibody and/or afunctional equivalent and/or a functional fragment thereof.

Other examples are Congo red or Thioflavin. The invention also disclosesa method for decreasing extracellular protein degradation and/or proteinclearance comprising decreasing an interaction between a compoundcomprising a cross-β structure and a compound comprising tPA-likeactivity, wherein the interaction is decreased by providing a compoundcapable of competing with the interaction. More in particular, thecompound capable of competing with the interaction comprises a fingerdomain and, even more particularly, the finger domain comprises astretch of at least 5 amino acid residues with alternating charge, forexample, ArgXGlu(X)₁₃Arg(X)₈GluXArg (SEQ ID NO: 1) or ArgXGluXLysXGluArg(SEQ ID NO: 2). In one aspect, the compound is fibronectin, FXII, HGFaor tPA.

In another embodiment, the invention also comprises a method forincreasing extracellular protein degradation and/or protein clearancecomprising increasing an interaction between a compound comprising across-β structure and a compound comprising tPA-like activity. This is,for example, accomplished by providing a compound capable of increasingan interaction between a compound comprising a cross-β structure and acompound comprising tPA-like activity. In one aspect, the compoundcapable of increasing an interaction between a compound comprising across-β structure and a compound comprising tPA-like activity is aprotein and/or a functional equivalent and/or a functional fragmentthereof. For example, an antibody which stabilizes the interactionbetween a compound comprising a cross-β structure and a compoundcomprising tPA-like activity, rendering the tPA-like activity in acontinuous activated state, results in increased protein degradationand/or protein clearance. However, it is appreciated that increasing aninteraction between a compound comprising a cross-β structure and acompound comprising tPA-like activity is also accomplished by mutationsin either the compound comprising a cross-β structure or in the compoundcomprising tPA-like activity, like swapping of domains (for example, byproviding the compound comprising tPA-like activity with other or morefinger domains obtainable from tPA, fibronectin, FXII or HGFa), or byincluding binding domains of, for example, RAGE or CD36.

In yet another embodiment, the invention discloses a method formodulating extracellular protein degradation and/or protein clearancecomprising modulating an interaction of a compound comprising tPA-likeactivity and the substrate of the activity. It is clear that there aremultiple ways by which the interaction can either be increased ordecreased. An increase in the interaction between a compound comprisingtPA-like activity and the substrate of the activity is, for example,accomplished by providing the compound comprising tPA-like activity witha mutation or mutations which improve the affinity of the compound withtPA-like activity for its substrate.

In yet another embodiment, the invention discloses a method for removingcross-β structures from the circulation, using a compound comprising across-β structure-binding domain. In one aspect, the compound is tPA orthe finger domain of tPA. It is clear that the invention also comprisesother cross-β structure-binding domains, including, but not limited to,the finger domains of HGFa, FXII and fibronectin (SEQ ID NOs: 3-17). Itis clear that the invention also comprises antibodies that bind cross-βstructures.

The present invention further discloses the use of a novel strategy toprevent the formation of, or to decrease/diminish, (amyloid) plaquesinvolved in a conformational disease, type II diabetes and/or aging(e.g., Alzheimer's disease). Plaques are typically defined asextracellular fibrillar protein deposits (fibrillar aggregates) and arecharacteristic of degenerative diseases. The “native” properties of theconstituent amyloid proteins may vary: some are soluble oligomers invivo (e.g., transthyretin in familial amyloid polyneuropathy), whereasothers are flexible peptides (e.g., amyloid-b in Alzheimer's disease(AD)). The basic pathogenesis of conformational diseases, for example,neurodegenerative disorders (AD, prion disorders), is thought to berelated to abnormal pathologic protein conformation, i.e., theconversion of a normal cellular and/or circulating protein into aninsoluble, aggregated, β-structure-rich form which is deposited in thebrain. These deposits are toxic and produce neuronal dysfunction anddeath. The formation of cross-β structures has thus far only beenassociated with severe pathological disorders. The results herein showthat tPA and other receptors for cross-β structure-forming proteins canbind denatured proteins, indicating that a large number of proteins arecapable of adopting a conformation containing cross-β or cross-β-likestructures. Taken together, the formation of a cross-β structureinitiates or participates in a physiological cascade of events necessaryto adequately deal with removal of unwanted molecules, i.e., misfoldedproteins, apoptotic cells or even pathogens. By increasing cross-βstructure formation in a protein involved in a conformational disease,the pathway for protein degradation and/or protein clearance isactivated and the protein is degraded, resulting in a decreasing plaqueor, in another aspect, the plaque is completely removed. Hence, theeffects of the conformational disease are diminished or, alternatively,completely abolished.

In a further embodiment, the invention discloses the use of a compoundcapable of increasing cross-β structure formation for diminishingplaques involved in a conformational disease. In another embodiment, theinvention discloses the use of a compound capable of binding to across-β structure for diminishing plaques and/or inhibiting cross-βstructure-mediated toxicity involved in a conformational disease. In oneuse of the invention, the compound is a protein and/or a functionalequivalent and/or a functional fragment thereof and, in another aspect,the protein is tPA, a finger domain, an antibody and/or a functionalequivalent and/or a functional fragment thereof. Examples of suchantibodies are 4B5 or 3H7.

In a yet further embodiment, the invention discloses the use of acompound capable of increasing tPA-like activity for diminishing plaquesinvolved in a conformational disease. In one aspect, the tPA-likeactivity is modified such that it has a higher activity or affinitytowards its substrate and/or cofactor. This is, for example,accomplished by providing the tPA-like activity with multiple bindingdomains for cross-β structure-comprising proteins. In another aspect,the binding domain comprises a finger domain and, in an additionalaspect, the finger domain comprises a stretch of at least five aminoacid residues with alternating charge, for exampleArgXGlu(x)₁₃Arg(X)₈GluXArg (SEQ ID NO: 1) or ArgXGluXLysXGluArg (SEQ IDNO: 2). In an additional embodiment, the finger domain is derived fromfibronectin, FXII, HGFa or tPA.

In yet another embodiment, the invention discloses the use of a compoundcapable of binding to a cross-β structure for the removal of cross-βstructures. In one aspect, the compound is a protein and/or a functionalequivalent and/or a functional fragment thereof. In an additionalaspect, the compound comprises tPA or tPA-like activity and/or afunctional equivalent and/or a functional fragment thereof. In a furtherembodiment, the functional fragment comprises a finger domain. In oneembodiment, the finger domain comprises a stretch of at least five aminoacid residues with alternating charge, for example,ArgXGlu(X)₁₃Arg(X)₈GluXArg (SEQ ID NO: 1) or ArgXGluXLysXGluArg (SEQ IDNO: 2). In yet an additional embodiment, the finger domain is derivedfrom fibronectin, FXII, HGFa or tPA. In another embodiment, the proteinis an antibody and/or a functional equivalent and/or a functionalfragment thereof. With this use, the invention discloses, for example, atherapeutic method to remove cross-β structure-comprising proteins from,for example, the circulation, such as via extracorporeal dialysis. Forexample, a patient with sepsis is subjected to such use by dialysis ofthe blood of that patient through means which are provided with, forexample, immobilized finger domains. One could, for example, couple thefinger domains to a carrier or to the inside of the tubes used fordialysis. In this way, all cross-β structure-comprising proteins will beremoved from the blood stream of the patient, thus, relieving patientsof the negative effects caused by the cross-β structure-comprisingproteins. Besides finger domain-comprising compounds, it is alsopossible to use other cross-β structure-binding compounds, likeantibodies or Congo Red. It is also clear that the use could be appliedin hemodialysis of kidney patients.

In yet another embodiment, the invention discloses the use of a compoundcapable of increasing or stabilizing an interaction of a compoundcomprising a cross-β structure and a compound comprising tPA-likeactivity for diminishing plaques involved in a conformational disease.Examples of a compound capable of increasing or stabilizing aninteraction of a compound comprising a cross-β structure and a compoundcomprising tPA-like activity are given herein. In another use, theinvention is used to treat the conformational disease Alzheimer ordiabetes. It is clear that the invention not only discloses a use todecrease/diminish plaques involved in a conformational disease, but alsothat the onset of the disease can also be inhibited or even completelyprevented. Examples of diseases which can be prevented and/or treatedaccording to the invention are conformational disease, amyloidosis-typediseases, atherosclerosis, diabetes, bleeding, thrombosis, cancer,sepsis and other inflammatory diseases, Multiple Sclerosis, auto-immunediseases, disease associated with loss of memory or Parkinson and otherneuronal diseases (epilepsy).

In another embodiment, the invention discloses the use of an antibodycapable of recognizing a cross-β structure epitope for determining thepresence of plaque involved in a conformational disease. In yet anotherembodiment, the invention discloses the use of a cross-βstructure-binding domain (such as a finger domain from, for example,tPA) for determining the presence of a plaque involved in aconformational disease.

These uses of the invention provide a new diagnostic tool. It was notuntil the present invention that a universal b-structure epitope wasdisclosed and that a diagnostic assay could be based on the presence ofthe cross-β structure. Such use is particularly useful for diagnosticidentification of conformational diseases or diseases associated withamyloid formation, such as Alzheimer or diabetes. It is clear that thisdiagnostic use is also useful for other diseases which involve cross-βstructure formation, like all amyloidosis-type diseases,atherosclerosis, diabetes, bleeding, cancer, sepsis and otherinflammatory diseases, Multiple Sclerosis, auto-immune diseases, diseaseassociated with loss of memory or Parkinsons and other neuronal diseases(epilepsy). For example, one can use a finger domain (of, for example,tPA) and provide it with a label (radioactive, fluorescent, etc.). Thislabeled finger domain may be used either in vitro or in vivo for thedetection of cross-β structure-comprising proteins and, thus, fordetermining the presence of a plaque involved in a conformationaldisease. One can, for example, use an ELISA assay to determine theamount of sepsis in a patient or one can localize a plaque involved in aconformational disease.

In yet another embodiment, the invention discloses a recombinant tPAcomprising an improved cross-β structure-binding domain or multiplecross-β structure-binding domains. In one aspect, tPA is provided withmultiple, possibly different, finger domains. A recombinant tPAcomprising an improved cross-β structure-binding domain or multiplecross-β structure-binding domains is used for different purposes, forexample, in a method for the improved treatment of thrombolysis or forthe removal of cross-β structure-comprising proteins from thecirculation of a patient in need thereof. Another use of a recombinanttPA comprising an improved cross-β structure-binding domain or multiplecross-β structure-binding domains is in diagnostic assays such as, forexample, in a BSE detection kit or in imaging experiments. This imagingwith a recombinant tPA comprising an improved cross-β structure-bindingdomain or multiple cross-β structure-binding domains is, for example,useful for detection of apoptosis. For example, labeled tPA, such as,but not limited to, radio-labeled tPA, is inoculated in an individual,followed by detection and localization of labeled tPA in the body. It isclear that recombinant tPA comprising a cross-β structure-binding domainor multiple cross-β structure-binding domains are also useful intherapeutic applications.

Because this invention has made clear that the cross-β structure isharmful when present in certain parts of the body, like the brain, forexample, and the damage is effected by the combination of cross-βstructures with tPA, a method is provided to inhibit cross-βstructure-mediated effects comprising providing an effective amount of aprotein comprising a finger domain to block the binding sites of thecross-β structure for tPA. Cross-β structure-mediated effects may evenbe further diminished by providing an effective amount of B-typecarboxypeptidase activity to inhibit the tPA activity.

In another embodiment, the local cross-β structure-mediated effect canbe used against tumors. To that effect, cross-β structure-mediatedeffects are locally induced to increase local cytotoxicity and/orfibrinolysis comprising locally administering an effective amount ofcross-β structures and/or cross-β structure-inducing compounds inconjunction with tPA or a compound with tPA-like activity and/or CPI ora compound with CPI-like activity.

The present invention discloses in a further embodiment a method whichis carried out ex vivo, e.g., by dialysis. According to this embodiment,the circulating fluid (blood) of a subject is brought in a systemoutside the body for clearing cross-β structures from the circulation.In one aspect, such a system is a flow-through system connected to thebody circulation with an inlet and an outlet. The cross-β structures arecleared by binding to a cross-β binding compound as definedhereinbefore. It is very important that no elements, such as the cross-βbinding compounds from the system, are brought into the subject'scirculation. For that reason, among others, preferred systems aredialysis systems. The invention further discloses devices for carryingout methods as disclosed herein. Thus, the invention discloses aseparation device for carrying out a method according to the inventionwherein the apparatus comprises a system for transporting circulationfluids ex vivo, the system provided with means for connecting to asubject's circulation for entry into the system and return from thesystem to the subject's circulation, the system comprising a solidphase, the solid phase comprising at least one compound capable ofbinding cross-β structures. Compounds for binding cross-β structureshave been disclosed herein. In one aspect, the device is a dialysisapparatus.

The invention also provides for detection of cross-β structures insamples. Such samples may be tissue samples, biopsies and the like, bodyfluid samples, such as blood, serum, liquor, CSF, urine, and the like.The invention thus discloses a method for detecting cross-β structuresin a sample, comprising contacting the sample with a compound capable ofbinding cross-β structures, allowing for binding of cross-β structuresto the compound and detecting the complex formed through binding.

Cross-β binding compounds have been defined hereinbefore. Detection ofthe complex or one of its constituents can be done through anyconventional means involving antibodies or other specific bindingcompounds, further cross-β binding compounds, etc. Detection can bedirect such as by labeling the complex or a binding partner for thecomplex or its constituents, or even by measuring a change in a physicalor chemical parameter of the complex versus unbound material. It mayalso be indirect by further binding compounds provided with a label. Alabel may be a radioactive label, an enzyme, a fluorescent molecule,etc.

The invention further discloses devices for carrying out the diagnosticmethods. Thus, the invention discloses a diagnostic device for carryingout a method according to the invention, comprising a sample container,a means for contacting the sample with a cross-β binding compound, across-β binding compound and a means for detecting bound cross-βstructures. In one embodiment, the device comprises a means forseparating unbound cross-β structures from bound cross-β structureswhich can be typically done by providing the cross-β binding compoundson a solid phase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the “cross-β structure pathway.”The cross-β structure is found in a number of proteins (1). Theformation of a cross-β structure can be triggered by severalphysiological or pathological conditions and subsequently initiates acascade of events, the “cross-β structure pathway.” Among the factorsthat trigger or regulate the formation of a cross-β structure within agiven protein are: 1) the physicochemical properties of the protein, 2)proteolysis, 3) regulated post-translational modification, includingcross-linking, oxidation, phosphorylation, glycosylation and glycation,4) glucose, and 5) zinc. Certain mutations within the sequence of aprotein are known to increase the ability of the protein to adopt across-β structure and form amyloid fibrils. These mutations are oftenfound in hereditary forms of amyloidosis, for example in AD. The presentinvention discloses multiple novel examples of proteins capable ofadopting a cross-β structure. Several proteins are known to bindcross-β-containing proteins (2). These proteins are part of the hereindisclosed signaling cascade (“cross-β structure pathway”) that istriggered upon formation of a cross-β structure. The “cross-β structurepathway” is modulated in many ways (3, 4, 5). Factors that regulate thepathway include modulators of synthesis and secretion including NOregulators, as well as modulators of activity, including proteaseinhibitors. The pathway is involved in many physiological andpathological processes including, but not limited to, atherosclerosis,diabetes, amyloidosis, bleeding, inflammation, multiple sclerosis,Parkinson's disease, sepsis, hemolytic uremic syndrome (7). Given theestablished role for tPA in long term potentiation, the “cross-βstructure pathway” may also be involved in learning.

FIG. 2 illustrates a cross-β structure in fibrin. Panel A depictsThioflavin T fluorescence of a fibrin clot. A fibrin clot was formed inthe presence of Thioflavin T and fluorescence was recorded at indicatedtime points. Background fluorescence of buffer, Thioflavin T and a clotformed in the absence of Thioflavin T, was substracted. Panel B is agraph depicting circular dichroism analysis of fibrin-derived peptides85, 86 and 87. Ellipticity (Dg.cm²/dmol) is plotted against wavelength(nm). The CD spectra demonstrates that peptides 85 and 86, but notpeptide 87, contain β-sheets. Panel C shows that X-ray fiber diffractionanalysis of peptide 85 reveals that the peptide forms cross-β sheets.Panel D is a graph showing plasminogen activation assay with fibrinpeptides 85, 86 and 87. It is seen that peptides 85 and 86, bothcontaining a cross-β structure, stimulate the formation of plasmin bytPA, whereas peptide 87, which lacks a cross-β structure, does not.

FIG. 3 is a set of graphs depicting binding of tPA, plasminogen andplasmin to Aβ. Aβ was coated onto plastic 96-well plates. Increasingconcentrations of either (A) tPA or (B) plasmin(ogen) were allowed tobind to the immobilized peptide. After extensive washing, tPA andplasmin(ogen) binding was assessed by enzyme-linked immunosorbent assaysusing anti-tPA and anti-plasminogen antibodies. Binding of (C) tPA and(D) plasmin to Aβ in the presence of 50 mM ε-aminocaproic acid (ε-ACA)was assessed as in A and B.

FIG. 4 is a set of graphs illustrating stimulation of tPA-mediatedplasmin formation by Aβ and synergistic stimulation of cell detachmentby plasminogen and Aβ. Panel A depicts that plasminogen (200 μg/ml) andtPA (200 pM) were incubated with Aβ (5 μM) or control buffer. Sampleswere taken from the reaction mixture at the indicated periods of timeand plasmin activity was measured by conversion of the chromogenicplasmin substrate S-2251 at 405 nm. Panel B shows that N1E-115 cellswere differentiated and received the indicated concentrations of plasminin the presence or absence of 25 μM Aβ. After 48 hours, the dead cellswere washed away and the remaining adherent cells were stained withmethylene blue. Plasmin cannot prevent Aβ-induced cell detachment. PanelC illustrates that N1E-115 cells were differentiated and received theindicated concentrations of plasminogen in the presence or absence of 10μM Aβ. After 24 hours, cell detachment was assessed. Aβ or plasminogenalone does not affect cell adhesion, but cause massive cell detachmentwhen added together. Panel D is an immunoblot analysis of plasminformation and laminin degradation. Differentiated N1E-115 cells weretreated with or without Aβ (10 μM) in the absence or presence of addedplasminogen. Addition of Aβ results in the formation of plasmin (bottompanel) and in degradation of laminin (top panel).

FIG. 5 depicts graphs illustrating that carboxypeptidase B inhibitsAβ-stimulated tPA-mediated plasmin formation and cell detachment. PanelA shows that plasminogen (200 μg/ml) and tPA (200 pM) were incubatedwith Aβ (5 μM) or control buffer. Samples were taken from the reactionmixture at the indicated periods of time and plasmin activity wasmeasured by conversion of the chromogenic plasmin substrate S-2251 at405 nm. The reaction was performed in the absence or the presence of 50μg ml⁻¹ carboxypeptidase B (CpB) and in the absence or presence of 3.5μM carboxypeptidase inhibitor (CPI). CpB greatly attenuates A-stimulatedplasmin formation. Panel B shows that N1E-115 cells were differentiatedand treated with Aβ (10 μM), plasminogen (Plg, 20 μg ml⁻¹) and/or CpB (1μM) as indicated. After 24 hours, the cells were photographed. Panel Cillustrates that, subsequently, the cells were washed once with PBS andthe remaining cells were quantified as percentage-adhered cells bymethylene blue staining. In Panel D, the cells were treated as in PanelsB and C and medium and cell fractions were collected and analyzed byWestern blot using an anti-plasmin(ogen) antibody. Aβ stimulates plasminformation that is inhibited by CpB.

FIG. 6 is a set of graphs illustrating that endostatin can form fibrilscomprising cross-β structure and stimulates plasminogen activation. InPanel A, TEM shows the formation of endostatin fibrils. Panel B containsan X-ray analysis that reveals the presence of cross-β structure inprecipitated (prec.) endostatin. Panel C is a plasminogen activationassay demonstrating the stimulating activity of cross-βstructure-containing endostatin on tPA-mediated plasmin formation. Aβ isshown for comparison. Panel D is an analysis of endostatin-induced celldeath by methylene blue staining. It is seen that only the precipitatedform is capable of efficiently inducing cell death. Direct cell death,but not cell detachment, is protected in the presence of sufficientglucose. Buffer prec. indicates control buffer.

FIG. 7 is a graph showing that IAPP stimulates tPA-mediated plasminogenactivation. Both full length (fl-hIAPP) and truncated amyloid core(Δ-hIAPP), but not mouse IAPP (Δ-mIAPP), stimulate tPA-mediatedplasminogen activation.

FIG. 8 is a set of graphs illustrating glycated albumin: Thioflavin Tand tPA binding, TEM images, X-ray fiber diffraction. Panel A is anELISA showing binding of tPA to albumin-g6p. Panel B shows competitionof tPA binding to albumin-g6p by Congo red as determined using ELISA.Panel C shows fluorescence measurements of Thioflavin T binding toalbumin-g6p, which is incubated for two, four, or 23 weeks. Panel Dshows that inhibition of the fluorescent signal is obtained uponincubation of 430 nM of albumin-g6p with 19 μM of Thioflavin T by tPA.Panels E and F illustrate that spectrophotometric analysis at 420 nmshows that increasing amounts of tPA result in a decrease of thespecific absorbance obtained upon incubation of 500 nM of albumin-g6pwith 10 μM of Thioflavin T. Panels G, H and I are electron micrographsshowing (G) amorphous precipitates of four weeks glycated albumin-g6p,(H) bundles of fibrillar aggregates of 23 weeks incubated albumin-g6p,and (I) two weeks glycated albumin-g6p. Panel J is an X-ray scatteringof albumin-g6p (23 weeks). Scattering intensities are color coded on alinear scale and decreases in the order white-grey-black. Scatteringfrom amorphous control albumin is substracted, as well as scatteringfrom the capillary glass wall and from air. d-spacings and the directionof the fiber axis are given and preferred orientations are indicatedwith arrows. Panel K is radial scan of albumin control and albumin-g6p(23 weeks). Panel L is a radial scan of albumin-g6p (23 weeks), showingrepeats originating from fibrous structure, after subtracting backgroundscattering of amorphous precipitated albumin. d-spacings (in Å) aredepicted above the peaks. Panel M contains tangential scans along the 20scattering-angles corresponding to indicate d-spacings. The scans showthat the 4.7 Å repeat, which corresponds to the hydrogen-bond distancewithin individual β-sheets, and the 6 Å repeat, are orientedperpendicular to the 2.3 Å repeat that runs parallel to the fiber axis.Panel N is a schematic drawing of the orientation of the cross-βstructures in albumin-g6p (23 weeks) amyloid fibrils.

FIG. 9 illustrates fibril formation of human hemoglobin. Panel A depictsbinding of tPA to in vitro glycated Hb-g6p. Panel B is an electronmicrograph showing in vitro glycated Hb, which aggregates in anamorphous and fibrous manner.

FIG. 10 shows that amyloid properties of albumin-AGE are introducedirrespective of the carbohydrate or carbohydrate derivative used forglycation. Panels A-I illustrate Congo red fluorescence of air-driedalbumin preparations. Fluorescence was measured with albumin incubatedwith buffer (Panel A) or with buffer and NaCNBH₃ (Panel B), with amyloidcore peptide of human IAPP (Panel C), Aβ (Panel D), with albuminincubated with g6p (Panel E), glucose (Panel F), fructose (Panel G),glyceraldehyde (Panel H), and glyoxylic acid (Panel I). Panel J showsthat Thioflavin T—amyloid fluorescence was measured in solution with theindicated albumin preparations. Panels K and L show that binding ofamyloid-binding serine protease tPA to albumin preparations was assayedusing an ELISA set-up. In Panel K, binding of tPA to albumin-glucose,-fructose, -glyceraldehyde, -glyoxylic acid, and albumin-buffer controlsis shown. In Panel L, binding of tPA to positive controls albumin-g6p,Aβ and IAPP is shown, as well as to albumin incubated with controlbuffer.

FIG. 11 illustrates analysis of Congo red and tPA binding to Aβ. Panel Ashows binding of tPA to immobilized Aβ as measured using an ELISA. PanelB illustrates the influence of increasing concentrations of Congo red onbinding of tPA to Aβ. In the ELISA, 10 μg ml⁻¹ of Aβ (1-40) was coatedand incubated with 40 nM of tPA and 0-100 μM of Congo red.

FIG. 12 illustrates binding of human FXII to amyloid peptides andproteins that contain the cross-β structure fold. Panels A and B showbinding of FXII to prototype amyloid peptides hAβ (1-40) and humanfibrin fragment α₁₄₇₋₁₅₉ FP13, and albumin-AGE and Hb-AGE, that allcontain cross-β structure, were tested in an ELISA. FXII does not bindto negative controls mouse Δ islet amyloid polypeptide (αmIAPP),albumin-control and Hb-control, all three lacking the amyloid-specificstructure. k_(D)'s for hAβ (1-40), FP13, albumin-AGE and Hb-AGE areapproximately 2, 11, 8 and 0.5 nM, respectively. Panels C and D depictthat coated hAβ (1-40) was incubated with 2.5 nM FXII in binding buffer,in the presence of a concentration series of human recombinanttissue-type plasminogen activator (ACTILYSE®, full-length tPA), orRETEPLASE® (K2P-tPA). The f.l. tPA- and K2P-tPA concentration was, atmaximum, 135 times the k_(D) for tPA binding to hAβ (1-40) (50 mM).Panels E and F show that coated amyloid albumin-AGE was incubated with15 nM FXII in binding buffer, in the presence of a concentration seriesof f.l. tPA or K2P-tPA. The tPA concentration was, at maximum, 150 timesthe k_(D) for tPA binding to albumin-AGE (1 nM). Panel G illustratesthat binding of FXII to hAβ (1-40) and the prototype amyloid humanamylin fragment hΔIAPP was tested using dot blot analysis. 10 μg of thepeptides that contain cross-β structure as well as the negative controlpeptide mΔIAPP and phosphate-buffered saline (PBS) were spotted induplicate. FXII specifically bound to hAβ (1-40) as well as to hΔIAPP.

FIG. 13 illustrates that finger domains bind to amyloid (poly)peptides.Panel A depicts binding of tPA and K2-P tPA to albumin-g6p. Panel Bshows binding of tPA and K2-P tPA to Aβ (1-40). The tPA antibody usedfor detection recognizes both tPA and K2-P-tPA with equal affinity (notshown). Panel C shows binding of tPA-F-GST and tPA to immobilized Aβ(1-40) and albumin-g6p. Control RPTPμ-GST does not bind Aβ oralbumin-g6p. Panel D is a pull-down assay with insoluble Aβ fibrils andtPA domains. Conditioned BHK medium from stably transfected cell linesexpressing tPA F, F-EGF, EGF, F-EGF-K1 and K1 with a C-terminal GST tag,as well as the tag alone, was used. “Control,” medium before thepull-down, “Aβ,” washed amyloid Aβ pellet, after the pull-down, “Sup,”medium after extraction with Aβ. Samples were analyzed on Western blotusing rabbit anti-GST antibody Z-5. Panels E-G are ELISA showing bindingof tPA F-EGF-GST and f.l. recombinant tPA to amyloid Aβ (Panel E), FP13(Panel F) and IAPP (Panel G). mΔIAPP was coated as non-amyloid negativecontrol (Panel E). Peptides were immobilized on ELISA plates andoverlayed with concentration series of tPA and F-EGF-GST. GST was usedas a negative control. Binding was detected using rabbit anti-GSTantibody Z-5. Panels H-M depict immunohistochemical analysis of bindingof tPA F-EGF-GST to amyloid deposits in human brain inflicted by AD.Brain sections were overlayed with tPA F-EGF-GST (Panels H and J) ornegative control GST (Panel L). The same sections were incubated withCongo red (Panels I, K and M) to locate amyloid deposits. Panels N and Oare pull-down assays with insoluble Aβ fibrils and finger domains.Recombinant F domains with a C-terminal GST tag were expressed by stablytransfected BHK cells. “Control,” medium before the pull-down, “Aβ,”washed amyloid Aβ pellet, after the pull-down, “Sup,” medium afterextraction with Aβ. Samples were analyzed on Western blot using rabbitanti-GST antibody Z-5.

FIG. 14 illustrates the finger module. Panel A is a schematicrepresentation of the location of the finger domain in tPA, factor XII,HGFa and fibronectin. Panel B is an alignment of the amino acid sequenceof the finger domain of the respective proteins. Specifically: tPA,FXII, HGFa, FN1-1, FN1-2, FN1-3, FN1-4, FN1-5, FN1-6, FN1-7, FN1-8,FN1-9, FN1-10, FN1-11, and FN1-12 (SEQ ID NOs: 3-17 respectively). PanelC is a representation of the peptide backbone of the tPA finger domainand the fourth and fifth finger domain of FN. Conserved disulfide bondsare shown in ball and stick.

FIG. 15 shows that antibodies elicited against amyloid peptidescross-react with glycated proteins, and vice versa. Panels A-C are ELISAwith immobilized g6p-glycated albumin-AGE:23 and Hb-AGE, theirnon-glycated controls (Panel A), Aβ (1-40) (Panel B), and IAPP andmΔIAPP (Panel C). For the Aβ ELISA, polyclonal anti-human vitronectinantibody α-hVn K9234 was used as a negative control. Panel D showsbinding of α-AGE1 to immobilized Aβ (1-40) on an ELISA plate afterpre-incubation of α-AGE 1 with IAPP fibrils. Panel E is a pull-downassay with anti-AGE1 antibody and amyloid fibrils of Aβ (16-22) (lanes1-2), Aβ (1-40) (lanes 4-5) and IAPP (lanes 6-7). After pelleting andwashing of the fibrils, samples were boiled in non-reducing samplebuffer and analyzed by SDS-PAGE. s=supernatant after amyloid extraction,p=amyloid pellet after extraction, m=molecular marker. Panels F and Gdepict that in an ELISA set-up, immobilized Aβ (1-40) (Panel F) and IAPP(Panel G) are co-incubated with tPA and 250 or 18 nM α-AGE1,respectively. Panel H shows that in an ELISA set-up binding of α-Aβ(1-42) H-43 to immobilized positive control Aβ (1-40), and to IAPP andalbumin-AGE:23 is tested. Albumin-control:23 and mΔIAPP are used asnegative controls. Panel I depicts binding of 100 nM α-Aβ (1-42) H-43 toIAPP, immobilized on an ELISA plate, in the presence of a concentrationseries of tPA. Panels J and K are ELISA showing binding of a polyclonalantibody in mouse serum elicited against albumin-AGE:23 and Aβ (1-40)(ratio 9:1) (“poab anti-amyloid”) and of a polyclonal antibody elicitedagainst a control protein (“control serum”) to immobilized IAPP (PanelJ) and albumin-AGE:23 (Panel K). Serum was diluted in PBS with 0.1% v/vTween 20. Panel L is an ELISA showing binding of mouse poab anti-amyloidserum to amyloid Aβ (1-40), hΔIAPP and fibrin fragment α₁₄₈₋₁₆₀ FP13.Control serum with antibodies raised against an unrelated protein,buffer and immobilized non-amyloid mΔIAPP and fibrin fragment α₁₄₈₋₁₅₇FP10 were used as negative controls. Panel M is an immunohistochemicalanalysis of the binding of rabbit anti-AGE2 to a spherical amyloidplaque (arrow) in a section of a human brain afflicted by AD.Magnification 400×. Panel N is a Congo red fluorescence of the samesection. Magnification 630×.

FIG. 16 illustrates that monoclonal anti-cross-β structure antibody 3H7detects glycated hemoglobin, Aβ, IAPP and FP13. ELISA showing binding ofmouse monoclonal anti-cross-β structure antibody 3H7 to (Panel A)glycated hemoglobin vs. control unglycated hemoglobin or (Panel B) Aβ,hIAPP, ΔmIAPP and fibrin fragment α₁₄₈₋₁₆₀ FP13.

FIG. 17 is a sandwich ELISA for detection of amyloid albumin-AGE oramyloid hemoglobin in solution. Immobilized recombinant tPA on Exiqonprotein Immobilizers was overlayed with albumin-AGE:23 solution oralbumin-control:23 solution at the indicated concentrations. Boundamyloid structures were detected with anti-Aβ (1-42) H-43 (A).

DETAILED DESCRIPTION OF THE INVENTION

The invention discloses (i) the identification of a “cross-β structurepathway,” (ii) the identification of multiligand receptors as beingcross-β structure receptors, (iii) the identification of the fingerdomain as a cross-β-binding module and (iv) the identification offinger-containing proteins, including tPA, FXII, HGFa and fibronectin aspart of the “cross-β structure pathway.”

This invention further discloses compounds not previously known to bindcross-β structure.

As disclosed herein, the invention describes compounds and methods forthe detection and treatment of diseases associated with the excessiveformation of a cross-β structure. Such diseases include knownconformational diseases including Alzheimer disease and other types ofamyloidosis. The present invention also discloses that other diseasesnot yet known to be associated with excessive formation of cross-βstructures are also caused by excessive formation of cross-β structures.Such diseases include atherosclerosis, sepsis, diffuse intravascularcoagulation, hemolytic uremic syndrome, preeclampsia, rheumatoidarthritis, autoimmune diseases, thrombosis and cancer.

According to the invention, the compound or means for binding thecross-β structure is a cross-β structure-binding molecule, such as afinger domain or a molecule containing one or more finger domains, or isa peptidomimetic analog of one or more finger domains. The compound canalso be an antibody or a functional fragment thereof directed to thecross-β structure.

According to the invention, the compound or means for binding thecross-β structure may also be a multiligand receptor or fragmentthereof. The compound may be, e.g., RAGE, CD36, Low density lipoproteinRelated Protein (LRP), Scavenger Receptor B-1 (SR-B1), SR-A, or afragment of one of these proteins.

The finger domains, finger-containing molecules or antibodies may behuman, mouse, rat or from any other species.

According to the invention, amino acids of the respective proteins maybe replaced by other amino acids which may increase/decrease theaffinity, the potency, bioavailability and/or half-life of the peptide.Alterations include conventional replacements (acid-acid, bulky-bulkyand the like), introducing D-amino acids, making peptides cyclic, etc.

Further, the invention discloses compounds and methods:

1) for detecting the presence of the cross-β structure;

2) for inhibiting the formation of amyloid fibrils;

3) for modulating cross-β structure-induced toxicity; and

4) for the removal of cross-β structure-containing molecules from thecirculation.

This invention also discloses methods for preparing an assay to measurecross-β structure in sample solutions.

This invention also discloses methods for detecting cross-β structure intissue samples or other samples obtained from living cells or animals.

This invention further discloses compounds and methods for preparing acomposition for inhibiting cross-β structure fibril formation.

This invention still further discloses compounds and methods forpreparing a composition for modulating cross-β structure-inducedtoxicity.

Abbreviations: Aβ, beta-amyloid peptide; AD, Alzheimer disease; AGE,advanced glycation end-products; CpB, carboxypeptidase B; COI(carboxypeptidase inhibitor); ELISA, enzyme-linked immunosorbent assay(ELISA); FN, fibronectin; FXII, factor XII (Hageman factor); HGFa,hepatocyte growth factor activator; IAPP, islet amyloid polypeptide;PCR, polymerase chain reactions (PCR); RAGE, receptor for AGE; tPA,tissue-type plasminogen activator.

The invention discloses compounds and methods for the detection andtreatment of diseases associated with the excessive formation of cross-βstructure.

The cross-β structure can be part of an Aβ fibril or part of anotheramyloid fibril. The cross-β structure can also be present in denaturedproteins.

The invention discloses methods to detect the cross-β structure. In oneembodiment, a cross-β structure-binding compound or means for bindingthe cross-β structure such as a finger domain or a molecule comprisingone or more finger modules, is bound or affixed to a solid surface, suchas a microtiter plate. The solid surfaces useful in this embodimentwould be known to one of skill in the art. For example, one embodimentof a solid surface is a bead, a column, a plastic dish, a plastic plate,a microscope slide, a nylon membrane, etc. After blocking, the surfaceis incubated with a sample. After removal of an unbound sample, boundmolecules comprising the cross-β structure are subsequently detectedusing a second cross-β structure-binding compound, such as ananti-cross-β structure antibody or a molecule containing a fingermodule. The second cross-β structure compound is bound to a label suchas an enzyme, i.e., peroxidase. The detectable label may also be afluorescent label, a biotin, a digoxigenin, a radioactive atom, aparamagnetic ion, and a chemiluminescent label. It may also be labeledby covalent means such as chemical, enzymatic or other appropriate meanswith a moiety such as an enzyme or radioisotope. Portions of theabove-mentioned compounds of the invention may be labeled by associationwith a detectable marker substance (e.g., radiolabeled with ¹²⁵I orbiotinylated) to provide reagents useful in detection and quantificationof a compound or its receptor-bearing cells or its derivatives in solidtissue, and fluid samples such as blood, cerebral spinal fluid, urine orothers. Such samples may also include serum used for tissue culture ormedium used for tissue culture.

In another embodiment, the solid surface can be microspheres, forexample, for agglutination tests.

In one embodiment, the compound containing a finger module is used tostain tissue samples. In one aspect, the compound or means for bindingthe cross-β structure is fused to a protein or peptide, such asglutathion-S-transferase. Alternatively, the compound is coupled to alabel. The detectable label may be a fluorescent label, a biotin, adigoxigenin, a radioactive atom, a paramagnetic ion, or achemiluminescent label. It may also be labeled by covalent means such aschemical, enzymatic or other appropriate means with a moiety such as anenzyme or radioisotope. Portions of the above-mentioned compounds of theinvention may be labeled by association with a detectable markersubstance (e.g., radiolabeled with ¹²⁵I, ^(99m)Tc, ¹³¹I, chelatedradiolabels, or biotinylated) to provide reagents useful in detectionand quantification of a compound or its receptor-bearing cells or itsderivatives in solid tissue, and fluid samples such as blood, cerebralspinal fluid or urine. The compound or means for binding the cross-βstructure is incubated with the sample and after washing, is visualizedwith antibodies directed against the fused protein or polypeptide, suchas glutathion-S-transferase.

In an embodiment, the sample is tissue from patients with or expected tosuffer from a conformational disease. Alternatively, the tissue isderived from animals or from cells cultured in vitro.

The methods of the invention disclose a new diagnostic tool. It was notuntil the present invention that a universal β-structure epitope wasdisclosed and that a diagnostic assay could be based on the presence ofthe cross-β structure. Such use is particularly useful for diagnosticidentification of conformational diseases or diseases associated withamyloid formation, such as Alzheimer or diabetes. It is clear that thisdiagnostic use is also useful for other diseases which involve cross-βstructure formation, like all amyloidosis-type diseases,atherosclerosis, diabetes, bleeding, cancer, sepsis and otherinflammatory diseases, Multiple Sclerosis, auto-immune diseases, diseaseassociated with loss of memory or Parkinson and other neuronal diseases(epilepsy). For example, one can use a finger domain (of, for example,tPA) and provide it with a label (radioactive, fluorescent, etc.). Thislabeled finger domain may be used either in vitro or in vivo for thedetection of cross-β structure-comprising proteins, hence, fordetermining the presence of a plaque involved in a conformationaldisease. One can use, for example, an ELISA assay to determine theamount of sepsis in a patient or one can localize a plaque involved in aconformational disease.

In another embodiment, this invention discloses a method for inhibitingthe formation of amyloid fibrils or to modulate cross-βstructure-induced toxicity. The compound is a cross-β-binding module,such as a finger domain, a finger domain-containing molecule, apeptidomimetic analog, and/or an anti-cross-β structure antibody, and/ora multiligand receptor or a fragment thereof.

According to the invention, the inhibition of fibril formation has theconsequence of decreasing the load of fibrils.

The inhibition of fibril formation or modulating cross-β structuretoxicity may also have the consequence of modulating cell death. Thecell can be any cell, but may be a neuronal cell, an endothelial cell,or a tumor cell. The cell can be a human cell or a cell from any otherspecies.

The cell may typically be present in a subject. The subject to which thecompound is administered may be a mammal or a human.

The subject may be suffering from amyloidosis, from anotherconformational disease, from prion disease, from chronic renal failureand/or dialysis-related amyloidosis, from atherosclerosis, fromcardiovascular disease, from autoimmune disease, or the subject may beobese. The subject may also be suffering from inflammation, rheumatoidarthritis, diabetes, retinopathy, sepsis, diffuse intravascularcoagulation, hemolytic uremic syndrome, and/or preeclampsia. Thediseases which may be treated or prevented with the methods of thepresent invention include, but are not limited to, diabetes, Alzheimerdisease, senility, renal failure, hyperlipidemic atherosclerosis,neuronal cytotoxicity, Down's syndrome, dementia associated with headtrauma, amyotrophic lateral sclerosis, multiple sclerosis, amyloidosis,an autoimmune disease, inflammation, a tumor, cancer, male impotence,wound healing, periodontal disease, neuropathy, retinopathy, nephropathyor neuronal degeneration.

The administration of compounds according to the invention may beconstant or for a certain period of time. The compound may be deliveredhourly, daily, weekly, monthly (e.g., in a time release form) or as aone time delivery. The delivery may also be continuous, e.g.,intravenous delivery.

A carrier may also be used to deliver the compound to a subject. Thecarrier may be a diluent, an aerosol, an aqueous solution, a non-aqueoussolution, or a solid carrier. This invention also disclosespharmaceutical compositions including therapeutically effective amountsof polypeptide compositions and compounds, together with suitablediluents, preservatives, solubilizers, emulsifiers, adjuvants and/orcarriers. Such compositions may be liquids or lyophilized or otherwisedried formulations and include diluents of various buffer content (e.g.,Tris-HCl., acetate, phosphate), pH and ionic strength, additives such asalbumin or gelatin to prevent absorption to surfaces, detergents (e.g.,Tween 20, Tween 80, Pluronic F68, bile acid salts), solubilizing agents(e.g., glycerol, polyethylene glycerol), antioxidants (e.g., ascorbicacid, sodium metabisulfite), preservatives (e.g., Thimerosal benzylalcohol, parabens), bulking substances or tonicity modifiers (e.g.,lactose, mannitol), covalent attachment of polymers such as polyethyleneglycol to the compound, complexation with metal ions, or incorporationof the compound into or onto particulate preparations of polymericcompounds such as polylactic acid, polglycolic acid, hydrogels, etc, oronto liposomes, microemulsions, micelles, uni-lamellar or multi-lamellarvesicles, erythrocyte ghosts, or spheroplasts.

The administration of compounds according to the invention may compriseintralesional, intraperitoneal, intramuscular or intravenous injection;infusion; liposome-mediated delivery; topical, intrathecal, gingivalpocket, per rectum, intrabronchial, nasal, oral, ocular or oticdelivery. In a further embodiment, the administration includesintrabronchial administration, anal, intrathecal administration ortransdermal delivery.

According to the invention, the compounds may be administered hourly,daily, weekly, monthly or annually. In another embodiment, the effectiveamount of the compound comprises from about 0.000001 mg/kg body weightto about 100 mg/kg body weight.

The compounds according to the invention may be delivered locally via acapsule which allows sustained release of the agent over a period oftime. Controlled or sustained release compositions include formulationin lipophilic depots (e.g., fatty acids, waxes, oils). Also included inthe invention are particulate compositions coated with polymers (e.g.,poloxamers or poloxalenes) and the agent coupled to antibodies directedagainst tissue-specific receptors, ligands or antigens or coupled toligands of tissue-specific receptors. Other embodiments of thecompositions of the invention incorporate particulate forms protectivecoatings, protease inhibitors or permeation enhancers for various routesof administration, including parenteral, pulmonary, nasal and/or oral.

The effective amount of the compounds according to the invention maycomprise 1 ng/kg body weight to about 1 gr/kg body weight. The actualeffective amount will be based upon the size of the compound and itsproperties.

The activity of tPA and/or the tPA-mediated activation of plasminogen isincreased by the binding to fibrin fragments or other protein fragmentsthat have a lysine or an arginine at the carboxy-terminal end. B-typecarboxypeptidases including, but not limited to, carboxypeptidase B(CpB) or Thrombin Activatable Fibrinolysis Inhibitor (TAFI, also namedcarboxypeptidase U or carboxypeptidase R), are enzymes that cleave offcarboxy-terminal lysine and arginine residues of fibrin fragments thatwould otherwise bind to tPA and/or plasminogen and stimulate plasminformation.

Because this invention has made clear that the cross-β structures areharmful when present in certain parts of the body like, for example, thebrain and the damage is effected by the combination of cross-βstructures with tPA, a method is disclosed to inhibit cross-βstructure-mediated effects comprising providing an effective amount of aprotein comprising a finger domain to block the binding sites of thecross-β structure for tPA. The cross-β structure-mediated effects mayeven be further diminished comprising providing an effective amount ofB-type carboxypeptidase activity to inhibit the tPA activity.

The invention discloses the use of a compound capable of binding to across-β structure for the removal of cross-β structures. The compound ormeans for binding the cross-β structure is a cross-β-binding molecule,such a protein and/or a functional equivalent and/or a functionalfragment thereof. In another aspect, the compound comprises a fingerdomain or a finger domain-containing molecule or a functional equivalentor a functional fragment thereof. In yet another aspect, the fingerdomain is derived from fibronectin, FXII, HGFa or tPA. It is clear thatthe invention also comprises antibodies that bind cross-β structures. Inanother embodiment, the protein is an antibody and/or a functionalequivalent and/or a functional fragment thereof. With this use, theinvention discloses, for example, a therapeutic method to remove cross-βstructure-comprising proteins from, for example, the circulation, suchas via extracorporeal dialysis. For example, a patient with sepsis issubjected to such use by dialysis of blood of the patient through meanswhich are provided with, for example, immobilized finger domains. Onecould, for example, couple the finger domains to a solid surface or tothe inside of the tubes used for dialysis. In this way, all cross-βstructure-comprising proteins will be removed from the bloodstream ofthe patient, thus, relieving the patient of the negative effects causedby the cross-β structure-comprising proteins. Besides fingerdomain-comprising compounds, it is also possible to use other cross-βstructure-binding compounds, like antibodies or soluble multiligandreceptors. It is also clear that the use could be applied inhemodialysis of kidney patients.

As used herein, “finger” encompasses a sequence that fulfills thecriteria outlined in FIG. 14. The sequence encompasses approximately 50amino acids, containing four cysteine residues at distinct spacing. Inone aspect, the finger domains of tPA, FXII, HGFa or fibronectin areused (SEQ ID NOs: 3-17). Alternatively, the “finger” may be apolypeptide analog or peptidomimetic with similar function, e.g., byhaving three-dimensional conformation. It is feasible that such analogshave improved properties.

EXAMPLES Example 1 Reagents

Bovine serum albumin (BSA) fraction V pH 7.0 and D-glucose-6-phosphatedi-sodium (g6p), D, L-glyceraldehyde, and chicken egg-white lysozymewere from ICN (Aurora, Ohio, USA). Rabbit anti-recombinant tissue-typeplasminogen activator (tPA) 385R and mouse anti-recombinant tPA 374Bwere purchased from American Diagnostica (Veenendaal, The Netherlands).Anti-laminin (L9393) was from Sigma. Swine anti-rabbitimmunoglobulins/HRP (SWARPO) and rabbit anti-mouse immunoglobulins/HRP(RAMPO) were from DAKO Diagnostics B.V. (The Netherlands). Alteplase(recombinant tissue-type plasminogen activator, tPA) was obtained fromBoehringer-Ingelheim (Germany). Reteplase (Rapilysin), a recombinantmutant tPA containing only kringle 2 and the catalytic domain (K2P-tPA)was obtained from Roche, Hertfordshire, UK, and porcine pancreascarboxypeptidase B (CpB) was from Roche, Mannheim, Germany.Carboxypeptidase inhibitor (CPI) was from Calbiochem (La Jolla, Calif.,USA). Tween 20 was purchased from Merck-Schuchardt (Hohenbrunn,Germany). Congo red was obtained from Aldrich (Milwaukee, Wisc., USA).Thioflavin T and lyophilized human hemoglobin (Hb) were from Sigma (St.Louis, Mo., USA). Lyophilized human fibrinogen was from Kordia (Leiden,The Netherlands). Chromogenic plasmin substrate S-2251 was purchasedfrom Chromogenix (Milan, Italy). Oligonucleotides were purchased fromSigma-Genosys (U.K.). Boro glass capillaries (0.5 mm Ø) were fromMueller (Berlin, Germany).

Example 2 Synthetic Peptides

Peptide Aβ (1-40), containing amino acids as present in the describedhuman Alzheimer peptide (DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV) (SEQID NO: 18), fibrin peptides 85 (or FP13) (KRLEVDIDIKIRS) (SEQ ID NO:19), 86 (or FP12) (KRLEVDIDIKIR) (SEQ ID NO: 20) and 87 (or FP10)(KRLEVDIDIK) (SEQ ID NO: 21), derived from the sequence of humanfibrin(ogen) and the islet amyloid polypeptide (IAPP) peptide orderivatives (fl-hIAPP: KCNTATCATQRLANFLVHSSNNFGAILSSTNVGSNTY (SEQ ID NO:22), ΔhIAPP (SNNFGAILSS) (SEQ ID NO:23 ), ΔmIAPP (SNNLGPVLPP) (SEQ IDNO: 24) were obtained from Pepscan, Inc. (The Netherlands) or from thepeptide synthesis facility at the Netherlands Cancer Institute (NCI,Amsterdam, The Netherlands). The peptides were dissolved in phosphatebuffered saline (PBS) to a final concentration of 1 mg ml⁻¹ and storedfor three weeks at room temperature (RT) to allow formation of fibrils.During this period, the suspension was vortexed twice weekly. Afterthree weeks, the suspension was stored at 4° C. Freeze-dried Aβ (1-40)from the NCI allowed to form cross-β structure in the same way. Cross-βstructure formation was followed in time by examination of Congo redbinding and green birefringence under polarized light.

Example 3 Congo Red Binding and Thioflavin T Fluorescence of a FibrinClot

For Thioflavin T-fluorescence measurements, 1 mg ml⁻¹ of fibrinogen wasincubated at 37° C. with 2 U ml⁻¹ of factor IIa in 150 mM NaCl, 20 mMTris-HCl pH 7.5, 10 mM CaCl₂, 50 μM Thioflavin T. Backgroundfluorescence of a clot was recorded in the absence of Thioflavin T andbackground Thioflavin T fluorescence was measured in the absence offactor IIa. Fluorescence was measured on a Hitachi F-4500 fluorescencespectrophotometer (Ltd., Tokyo, Japan), using Sarstedt REF67.754cuvettes. Apparatus settings: excitation at 435 nm (slit 10 nm),emission at 485 nm (slit 10 nm), PMT voltage 950 V, measuring time 10seconds, delay 0 seconds. For detection of Congo red binding, a fibrinclot was formed at room temperature as described above (Thioflavin T wasomitted in the buffer). The clot was incubated with Congo red solutionand washed according to the manufacturer's recommendations (SigmaDiagnostics, MO, USA). The clot was analyzed under polarized light.

Example 4 Initial Preparation of Glycated Albumin, Hemoglobin (Hb) andLysozyme

For preparation of advanced glycation end-product modified bovine serumalbumin (albumin-g6p), 100 mg ml⁻¹ of albumin was incubated with PBScontaining 1 M of g6p and 0.05% m/v NaN₃, at 37° C. in the dark. Onealbumin solution was glycated for two weeks, a different batch ofalbumin-was glycated for four weeks. Glycation was prolonged up to 23weeks with part of the latter batch. Human Hb at 5 mg ml⁻¹ was incubatedfor ten weeks at 37° C. with PBS containing 1 M of g6p and 0.05% m/v ofNaN₃. In Addition, a Hb solution of 50 mg ml⁻¹ was incubated for eightweeks with the same buffer. For preparation of glyceraldehyde-modifiedalbumin (albumin-glyceraldehyde) and chicken egg-white lysozyme(lysozyme-glyceraldehyde), filter-sterilized protein solutions of 15 mgml⁻¹ were incubated for two weeks with PBS containing 10 mM ofglyceraldehyde. In controls, g6p or glyceraldehyde was omitted in thesolutions. After incubations, albumin and lysozyme solutions wereextensively dialyzed against distilled water and subsequently stored at−20° C. Protein concentrations were determined with Advancedprotein-assay reagent ADV01 (Cytoskeleton, Denver, Colo., USA).Glycation was confirmed by measuring intrinsic fluorescent signals fromadvanced glycation end-products; excitation wavelength 380 nm, emissionwavelength 435 nm.

Example 5 Further Experiment Involving Glycation

For preparation of albumin-AGE, 100 mg ml⁻¹ bovine serum albumin(fraction V, catalogue # A-7906, initial fractionation by heat shock,purity≧98% (electrophoresis), remainder mostly globulins, Sigma-Aldrich,St. Louis, Mo., USA) was incubated at 37° C. in the dark, withphosphate-buffered saline (PBS, 140 mM sodium chloride, 2.7 mM potassiumchloride, 10 mM disodium hydrogen phosphate, 1.8 mM potassiumdi-hydrogen phosphate, pH 7.3), 1 M D-glucose-6-phosphate disodium salthydrate (anhydrous) (ICN, Aurora, Ohio, USA) and 0.055% (m/v) NaN₃.Bovine albumin has 83 potential glycation sites (59 lysine and 23arginine residues, N-terminus). Albumin was glycated for two weeks(albumin-AGE:2), four weeks (albumin-AGE:4) or 23 weeks(albumin-AGE:23). In controls, g6p was omitted. After incubation,solutions were extensively dialyzed against distilled water andsubsequently stored at 4° C. Protein concentrations were determined withadvanced protein-assay reagent ADV01 (Cytoskeleton, CO, USA).Alternatively, albumin was incubated for 86 weeks with 1 M g6p, 250 mMDL-glyceraldehyde (ICN, Aurora, Ohio, USA)/100 mM NaCNBH₃, 1 Mβ-D-(−)-fructose (ICN, Aurora, Ohio, USA), 1 M D(+)-glucose (BDH, Poole,England), 500 mM glyoxylic acid monohydrate (ICN, Aurora, Ohio, USA)/100mM NaCNBH₃, and corresponding PBS and PBS/NaCNBH₃ buffer controls.Glycation was confirmed (i) by observation of intense brown staining,(ii) by the presence of multimers on SDS-polyacrylamide gels, (iii) byassaying binding of AGE-specific antibodies moab anti-albumin-g6p 4B5⁴⁶and poab anti-fibronectin-g6p (Ph. De Groot/I. Bobbink, UMC Utrecht;unpublished data), and (iv) by measuring intrinsic fluorescent signalsfrom AGE (excitation wavelength 380 nm, emission wavelength 445 nm).Autofluorescent signals of albumin controls were less than 4% of thesignals measured for albumin-AGE and were used for backgroundcorrections.

Example 6 Isolation of Hb from Human Erythrocytes

Human Hb was isolated from erythrocytes in EDTA-anticoagulated blood ofthree healthy individuals and of 16 diabetic patients. 100 μl of wholeblood was diluted in 5 ml of physiological salt (154 mM NaCl), cellswere gently spun down, and resuspended in 5 ml of physiological salt.After 16 hours incubation at room temperature, cells were again spundown. Pelleted cells were lysed by adding 2 ml of 0.1 M of boric acid,pH 6.5 and subsequently, cell debris was spun down. Supernatant wascollected and stored at −20° C.

Example 7 Determination of GlycoHB Concentrations

Concentrations of glycated Hb, also named glycohemoglobin, or namedHb_(Alc,) in EDTA-blood of human healthy donors or diabetic patients,were determined using a turbidimetric inhibition immunoassay withhemolyzed whole blood, according to the manufacturer's recommendations(Roche Diagnostics, Mannheim, Germany). Standard deviations are 2.3% ofthe measured Hb_(AlC) concentrations.

Example 8 Binding of Congo Red to Glycated Albumin

Binding of Congo red to albumin-AGE glycated for 86 weeks withcarbohydrates glucose, fructose and glucose-6-phosphate, or withcarbohydrate derivatives glyceraldehyde and glyoxylic acid, was testedusing air-dried samples. For this purpose, 5 μg albumin was applied to aglass cover slip and air dried. Samples were incubated with Congo redand subsequently washed according to the manufacturer's recommendations(Sigma Diagnostics, St Louis, Mo., USA). Pictures were taken on a LeicaDMIRBE fluorescence microscope (Rijswijk, The Netherlands) using 596 nmand 620 nm excitation and emission wavelengths, respectively.

Example 9 Endostatin Preparations

Endostatin was purified from Escherichia coli essentially asdescribed.⁴⁷ In short, B121.DE3 bacteria expressing endostatin werelysed in a buffer containing 8 M urea, 10 mM Tris (pH 8.0), 10 mMimidazole and 10 mM β-mercapto-ethanol. Following purification overNi²⁺-agarose, the protein sample was extensively dialyzed against H₂O.During dialysis, endostatin precipitates as a fine white solid. Aliquotsof this material were either stored at −80° C. for later use, or werefreeze-dried prior to storage. Soluble endostatin produced in the yeaststrain Pichia pastoris was kindly provided by Dr. Kim Lee Sim (EntreMed,Inc., Rockville, Mass., USA). Aggregated endostatin was prepared fromsoluble endostatin as follows. Soluble yeast endostatin was dialyzedovernight in 8 M urea and subsequently three times against H₂O. Likebacterial endostatin, yeast endostatin precipitates as a fine whitesolid.

Example 10 Congo Red Staining

Freeze-dried bacterial endostatin was resuspended in either 0.1% formicacid (FA) or in dimethyl-sulfoxide and taken up in a glass capillary.The solvent was allowed to evaporate and the resulting endostatinmaterial was stained with Congo red according to the manufacturer'sprotocol (Sigma Diagnostics, St. Louis, Mo., USA).

Example 11 Circular Dichroism Measurements

UV circular dichroism (CD) spectra of peptide and protein solutions (100μg ml⁻¹ in H₂O) were measured on a JASCO J-810 CD spectropolarimeter(Tokyo, Japan). Averaged absorption spectra of five or ten singlemeasurements from 190-240 nm or from 190-250 nm, for fibrin peptides 85,86, 87 or for albumin, glycated albumin and human Aβ (16-22),respectively, are recorded. The CD spectrum of Aβ (16-22) was measuredas a positive control. Aβ (16-22) readily adopts amyloid fibrilconformation with cross-β structure when incubated in H₂O⁴⁵. For albuminand Aβ (16-22), relative percentage of the secondary structure elementspresent was estimated using k2d software.⁴⁸

Example 12 X-Ray Fiber Diffraction

Aggregated endostatin was solubilized in 0.1% FA, lyophilized fibrinpeptides were dissolved in H₂O and glycated albumin was extensivelydialyzed against water. Samples were taken up in a glass capillary. Thesolvent was allowed to evaporate over a period of several days.Capillaries containing the dried samples were placed on a NoniuskappaCCD diffractometer (Bruker-Nonius, Delft, The Netherlands).Scattering was measured using sealed tube MoKα radiation with a graphitemonochromator on the CCD area detector during 16 hours. Scattering fromair and the glass capillary wall were subtracted using in-house software(VIEW/EVAL, Dept. of Crystal- and Structural Chemistry, UtrechtUniversity, The Netherlands).

Example 13 Transmission Electron Microscopy

Endostatin, hemoglobin and albumin samples were applied to 400 meshspecimen grids covered with carbon-coated collodion films. After fiveminutes, the drops were removed with filter paper and the preparationswere stained with 1% methylcellulose and 1% uranyl acetate. Afterwashing in H₂O, the samples were dehydrated in a graded series of EtOHand hexanethyldisilazane. Transmission electron microscopy (TEM) imageswere recorded at 60 kV on a JEM-1200EX electron microscope (JEOL,Japan).

Example 14 Enzyme-Linked Immunosorbent Assay: Binding of tPA to GlycatedAlbumin, Hb and Aβ (1-40)

Binding of tPA to albumin-g6p (four-week and 23-week incubations),albumin-glyceraldehyde, control albumin, human Hb-g6p (ten-weekincubation), Hb control, or to Aβ (1-40) was tested using anenzyme-linked immunosorbent assay (ELISA) set-up. Albumin-g6p andcontrol albumin (2.5 μg ml⁻¹ in coat buffer, 50 mM Na₂CO₃/NaHCO₃ pH 9.6,0.02% m/v NaN₃, 50 μl/well) were immobilized for one hour at roomtemperature in 96-well protein Immobilizer plates (Exiqon, Vedbaek,Denmark). Aβ (1-40) (10 μg ml⁻¹ in coat buffer) was immobilized for 75minutes at room temperature in a 96-well peptide Immobilizer plate(Exiqon, Vedbaek, Denmark). Control wells were incubated with coatbuffer only. After a wash step with 200 μl of PBS/0.1% v/v Tween 20,plates were blocked with 300 μl of PBS/1% v/v Tween 20, for two hours atroom temperature while shaking. All subsequent incubations wereperformed in PBS/0.1% v/v Tween 20 for one hour at room temperaturewhile shaking, with volumes of 50 μl per well. After each incubation,wells were washed five times with 200 μl of PBS/0.1% v/v Tween 20.Increasing amounts of f.l. tPA or K2-P tPA was added in triplicate tocoated wells and to control wells. Antibody 385R and subsequentlySWARPO, or antibody 374B and subsequently RAMPO, were added to the wellsat a concentration of 1 μg ml⁻¹. Bound peroxidase-labeled antibody wasvisualized using 100 μl of a solution containing 8 mg ofortho-phenylene-diamine and 0.0175% v/v of H₂O₂ in 20 ml of 50 mM citricacid/100 mM Na₂HPO₄ pH 50. Staining was stopped upon adding 50 μl of a2-M H₂SO₄ solution. Absorbance was read at 490 nm on a V_(max) kineticmicroplate reader (Molecular Devices, Sunnyvale, Calif., USA).

Competition experiments were performed with 20 or 40 nM of tPA with,respectively, albumin-g6p or Aβ (1-40) and with increasing amounts ofCongo red in PBS/0.08% v/v Tween 20/2% v/v EtOH.

Example 15 ELISA: Binding of tPA to Albumin-AGE

Binding of the cross-B structure marker tPA to albumin-AGE was testedusing an ELISA setup. We showed that tPA binds to prototype amyloidpeptides human Aβ (1-40) and human IAPP⁴⁹ (this application). Therefore,we used tPA binding to these two peptides as positive control. The86-week glycated samples and controls were coated to Greiner microlonplates (catalogue # 655092, Greiner, Frickenhausen, Germany). Wells wereblocked with Superblock (Pierce, Rockford, Ill., USA). All subsequentincubations were performed in PBS/0.1% (v/v) Tween 20 for one hour atroom temperature while shaking, with volumes of 50 μl per well. Afterincubation, wells were washed five times with 300 μl PBS/0.1% (v/v)Tween 20. Increasing concentrations of tPA were added in triplicate tocoated wells as well as to control wells. During tPA incubations of86-week incubated samples, at least a 123,000 times molar excess ofε-amino caproic acid (εACA, 10 mM) was added to the solutions. εACA is alysine analogue and is used to avoid potential binding of tPA to albuminvia its kringle 2 domain.⁵⁰ Monoclonal antibody 374b (AmericanDiagnostica, Instrumentation laboratory, Breda, The Netherlands) and,subsequently, RAMPO (Dako diagnostics, Glostrup, Denmark) was added tothe wells at a concentration of 0.3 μg ml⁻¹. Bound peroxidase-labeledantibody was visualized using 100 μl of a solution containing 8 mgortho-phenylene-diamine in 20 ml 50 mM citric acid/100 mM Na₂HPO₄ pH 5.0with 0.0175% (v/v) H₂O₂. Staining was stopped upon adding 50 μl of a 2 MH₂SO₄ solution. Absorbance was read at 490 nm on a V_(max) kineticmicroplate reader (Molecular Devices, CA, USA). Background signals fromnon-coated control wells were substracted from corresponding coatedwells.

Example 16 Thioflavin T Fluorescence of Glycated Albumin and Lysozyme,and tPA

Initially, for fluorescence measurements, 500 nM of albumin-g6p,albumin-glyceraldehyde, control albumin, lysozyme-glyceraldehyde, orcontrol lysozyme were incubated with increasing amounts of Thioflavin T,in 50 mM of glycine-NaOH, pH 9. For blank readings, an identicalThioflavin T dilution range was prepared without protein or Thioflavin Twas omitted in the protein solutions. Samples were prepared intriplicate.

Example 17 Thioflavin T Fluorescence

In further experiments, fluorescence measurements with albumin-g6p;2,albumin-g6p:4, albumin-g6p:23 and controls in 50 mM glycine-NaOH, pH 9were incubated with increasing amounts of ThT (Sigma-Aldrich Chemie,Steinheim, Germany), a marker for amyloid cross-β structure.⁵¹Albumin-AGE:4 concentration was 175 nM; other protein concentrationswere 500 nM. For fluorescence measurements with 86-week glycatedsamples, 140 nM of protein was incubated with a fixed concentration of20 μM ThT. Fluorescence was measured in triplicate on a Hitachi F-4500fluorescence spectrophotometer (Ltd., Tokyo, Japan), after one hourincubation at room temperature. Excitation and emission wavelengths were435 nm (slit 10 nm) and 485 nm (slit 10 nm), respectively. Backgroundsignals from buffer and protein solutions without ThT were substractedfrom corresponding measurements with protein solution incubated withThT.

Example 18 Fluorescence: Competitive Binding of Thioflavin T and tPA toAlbumin-g6p

A solution of 430 nM albumin-g6p and 19 μM of Thioflavin T was incubatedwith increasing amounts of tPA, for one hour at room temperature. Forblank readings, albumin-g6p was omitted. Samples were prepared infour-fold in 50 mM glycine-NaOH pH 9. Emission measurements wereperformed as described above.

Example 19 Absorbance: Competitive Binding of Thioflavin T and tPA toAlbumin-g6p

Albumin-g6p (500 nM) and Thioflavin T (10 μM) were incubated withincreasing amounts of tPA, in 50 mM glycine-NaOH pH 9, for one hour atroom temperature. Absorbance measurements were performed at thealbumin-g6p Thioflavin T absorbance maximum at 420 nm. Samples wereprepared in four-fold. For blank readings, albumin-g6p was omitted inthe solutions. Absorbance was read in a quartz cuvette on a PharmaciaBiotech Ultrospec 3000 UV/visible spectrophotometer (Cambridge,England).

Example 20 Plasminogen Activation Assay

Plasminogen (200 μg ml⁻¹) was incubated with tPA (200 pM) in thepresence or the absence of a co-factor (5 μM of either endostatin, Aβ(1-40), or one the fibrin-derived peptides 85, 86 and 87). At theindicated time intervals, samples were taken and the reaction wasstopped in a buffer containing 5 mM EDTA and 150 mM εACA. Aftercollection of the samples, a chromogenic plasmin substrate S-2251 wasadded and plasmin activity was determined kinetically in aspectrophotometer at 37° C.

Example 21 N1E-115 Cell Culture and Differentiation

N1E-115 mouse neuroblastoma cells were routinely cultured in DMEMcontaining 5% FCS, supplemented with antibiotics. Cells weredifferentiated into post-mitotic neurons.⁵² The cells were exposed to Aβ(50 μg ml⁻¹) for 24 hours in the presence or absence of 20 μg ml⁻¹plasminogen in the presence or absence of 50 μg ml⁻¹ CpB. Cells werephotographed, counted and lysed by the addition of 4× sample buffer (250mM Tris pH 6.8, 8% SDS, 10% glycerol, 100 mM DTT, 0.01% w/v bromophenolblue) to the medium. The lysate, containing both adherent and floating(presumably dying and/or dead) cells, as well as the culture medium,were analyzed for the presence of plasminogen and plasmin, as well asfor laminin, by Western blot analysis using specific antibodies againstplasminogen (MoAb 3642, American Diagnostics), laminin (PoAb L9393,Sigma).

Example 22 Binding of Human Factor XII to Amyloid Peptides and Proteinsthat Contain the Cross-β Structure Fold

The binding of human FXII (Calbiochem, La Jolla, Calif., USA, catalogue#233490) to amyloid (poly)peptides was tested. Prototype amyloidpeptides human amyloid-β (1-40) (hAβ (1-40)) and human fibrin fragmentα₁₄₇₋₁₅₉ FP13, and glucose-6-phosphate glycated bovine albumin(albumin-advanced glycation end product (AGE)) and glucose-6-phosphateglycated human hemoglobin (Hb-AGE), all containing cross-β structure, aswell as negative controls mouse Δ islet amyloid polypeptide (ΔmIAPP),albumin-control and Hb-control, all three lacking the amyloid-specificstructure, were coated to ELISA plates and overlayed with aconcentration series of human factor XII. Binding of FXII was detectedusing a rabbit polyclonal anti-FXII antibody (Calbiochem, La Jolla,Calif., USA, catalogue # 233504) and peroxidase-labeled swineanti-rabbit IgG. Wells were coated in triplicate. The FXII bindingbuffer included 10 mM HEPES pH 7.3, 137 mM NaCl, 11 mM D-glucose, 4 mMKCl, 1 mg ml⁻¹ albumin, 50 μM ZnCl₂, 0.02% (m/v) NaN₃ and 10 mM ε-aminocaproic acid (εACA). Lysine analogue εACA was added to avoid putativebinding of FXII to cross-β structure via the FXII kringle domain. Inaddition, binding of FXII to hAβ (1-40) and the prototype amyloid humanamylin fragment hΔIAPP was tested using dot blot analysis. 10 μg of thepeptides that contain cross-β structure, as well as the negative controlpeptide mΔIAPP and phosphate-buffered saline (PBS) were spotted induplicate onto methanol-activated nitrocellulose. Spots weresubsequently incubated with 2 nM FXII in FXII buffer or with FXII bufferalone, anti-FXII antibody, and SWARPO. Binding of FXII was visualized bychemiluminescence upon incubation with enhanced luminol reagent(PerkinElmer Life Sciences, Boston, Mass., USA). To test whether FXIIand tPA, which is known for its capacity to bind to polypeptides thatcontain the cross-β structure fold,⁴⁹ bind to overlapping binding siteson amyloid (poly)peptides, competitive ELISAs were performed. Coated hAβ(1-40) or amyloid albumin-AGE were incubated with 2.5 nM or 15 nM FXIIin binding buffer in the presence of a concentration series of humanrecombinant tissue-type plasminogen activator (ACTILYSE®, full-lengthtPA) or RETEPLASE® (K2P-tPA). RETEPLASE® is a truncated form of tPA thatincludes the second kringle domain and the protease domain. The f.l. tPAand K2P-tPA concentration was at maximum 135 times the k_(D) for tPAbinding to hAβ (1-40) (50 nM) or 150 times the k_(D) for tPA binding toalbumin-AGE (1 nM).

Example 23 Cloning Procedure

Cloning of the amino-terminal finger domain (F) of human tPA, residuesSer1-Ser50, preceded by the pro-peptide (residues Met-35-Arg-1) and aBglII restriction site, was performed by using PCR and standardrecombinant DNA techniques. In brief, the pro-peptide finger region wasamplified by PCR using 1 ng of plasmin Zp17,⁵³ containing the cDNAencoding tPA as a template. Oligonucleotides used were5′AAAAGTCGACAGCCGCCACCATGGATGCAATGAAGAGA (SEQ ID NO: 25) (1) and3′AAAAGCGGCCGCCCACTTTTGACAGGCACTGAG (SEQ ID NO: 26) (2) comprising aSalI or a NotI restriction site, respectively (underlined). The PCRproduct was cloned in a SalI/NotI-digested expression vector,pMT2-GST.⁵⁴ As a result, a construct is generated that contains a SalIrestriction site, the coding sequence for the finger domain of tPA, aNotI and a KpnI restriction site, a thrombin cleavage-site (TCS), aglutathion-S-transferase (GST) tag and an EcoRI restriction site. Theappropriate sequence of the construct was confirmed by sequenceanalysis. In a similar way, a construct consisting of the tPA F-EGFdomains was prepared. Next, the constructs were ligated SalI-EcoRI inpGEM3Zf(−) (Promega, Madison, Wisc., USA). The HindIII-SalI-tPApro-peptide-BglII-F-NotI-KpnI-TCS-GST-EcoRI construct was used as acloning cassette for preparation of constructs containing tPA K1,F-EGF-K1, EGF, as well as human hepatocyte growth factor activator F andF-EGF, human factor XII F and F-EGF, and human fibronectin F4, F5, F4-5and F10-12. Subsequently, constructs were ligated HindIII-EcoRI in thepcDNA3 expression vector (Invitrogen, Breda, The Netherlands). Inaddition, the GST tag alone was cloned into pcDNA3, preceded by the tPApro-peptide. Primers used for constructs were: tPA F-EGF (SEQ ID NO: 27)3′AAAAGCGGCCGCGTGGCCCTGGTATCTATTTC (3) and (1) tPA EGF (SEQ ID NO: 28)5′AAAAGAGATCTGTGCCTGTCAAAAGTTGC (4) and (2) tPA K1 (SEQ ID NO: 29)5′AAAAGAGATCTGATACCAGGGCCACGTGCTAC (5) (SEQ ID NO: 30)3′AAAAGCGGCCGCCCGTCACTGTTTCCCTCAGAGCA (6) tPA F-EGF-K1 (1) and (6) GSTtag (SEQ ID NO: 31) AAAAGCGGCCGCCTGGCTCCTCTTCTGAATC (1) and (7)Fibronectin F4 (SEQ ID NO: 32) 5′TGCAAGATCTATAGCTGAGAAGTGTTTTGAT (8)(SEQ ID NO: 33) 3′GATGCGGCCGCCCTGTATTCCTAGAAGTGCAAGTG (9) Fibronectin F5(SEQ ID NO: 34) 5′TGCAAGATCTACTTCTAGAAATAGATGCAAC (10) (SEQ ID NO: 35)3′TGATGCGGCCGCCCCACAGAGGTGTGCCTCTC (11) Fibronectin F4-5 (8) and (11)Fibronectin F10-12 (SEQ ID NO: 36) 5′AAAAAAGATCTAACCAACCTACGGATGACTC(12) (SEQ ID NO: 37) 3′AAAAAAGGTACCGACTGGGTTCACCCCCAGGT (13) factor XIIF (SEQ ID NO: 38) 5′GAAACAAGATCTCAGAAAGAGAAGTGCTTTGA (14) (SEQ ID NO:39) 3′ACGGGCGGCCGCCCGGCCTGGCTGGCCAGCCGCT (15) factor XII F-EGF (SEQ IDNO: 40) 5′AAAAAAGATCTCAGAAAGAGAAGTGCTTTGA (16) (SEQ ID NO: 41)3′AAAAAGGTACCGGCTTGCCTTGGTGTCCACG (17) HGFa F (SEQ ID NO: 42)5′GCAAGAAGATCTGGCACAGAGAAATGCTTTGA (18) (SEQ ID NO: 43)3′AAGGGCGGCCGCCCAGCTGTATGTCGGGTGCCTT (19) HGFa F-EGF (SEQ ID NO: 44)5′AAAAAAGATCTGGCACAGAGAATGCTTTGA (20) (SEQ ID NO: 45)3′AAAAAGGTACCGCTCATCAGGCTCGATGTTG (21)

Example 24 Transient Expression of tPA-F-GST in 293T Cells

Initially, 293T cells were grown in RPMI1640 medium (Invitrogen,Scotland, U.K.) supplemented with 5% v/v fetal calf-serum, penicillin,streptomycin and guanidine, to 15% confluency. Cells were transientlytransfected using Fugene-6, according to the manufacturer'srecommendations (Roche, Ind., USA). pMT2-tPA-F-GST containing the tPAfragment, or a control plasmid, pMT2-RPTPμ-GST, containing theextracellular domain of receptor-like protein tyrosine phosphatase [(RPTPμ)⁵⁴ were transfected, and medium was harvested after 48 hourstransfection. Expression of tPA-F-GST and RPTPμ-GST in 293T medium wasverified by immunoblotting. Collected samples were run out on SDS-PAAgels after the addition of 2× sample buffer. Gels were blotted onnitrocellulose membranes. Membranes were blocked in 1% milk (Nutricia)and incubated with primary monoclonal anti-GST antibody 2F3⁵⁴ andsecondary HRP-conjugated rabbit anti-mouse IgG (RAMPO). The blots weredeveloped using Western Lightning Chemiluminescence Reagent Plus(PerkinElmer Life Sciences, MA, USA).

Example 25 Stable Expression of Finger Constructs in BHK Cells

Baby hamster kidney cells were seeded in DMEM/NUT mix F-12 (HAM) medium(Invitrogen, U.K.) supplemented with 5% v/v fetal calf-serum (FCS),penicillin, streptomycin and guanidine, to 1% confluency. Cells werestably transfected by using the Ca₃(PO₄)₂—DNA precipitation method.After 24 hours, medium was supplemented with 1 mg ml⁻¹ geneticin G-418sulphate (Gibco, U.K.). Medium with G-418 was refreshed several timesduring ten days to remove dead cells. After this period of time, stablesingle colonies were transferred to new culture flasks and cells weregrown to confluency. Expression of constructs was verified byimmunoblotting. Collected samples were run out on SDS-PAA gels after theaddition of 2× sample buffer. Gels were blotted on nitrocellulosemembranes. Membranes were blocked in 5% milk (Nutricia) with 1.5% mn/vBSA and incubated with primary monoclonal anti-GST antibody (Santa CruzBiotechnology, Santa Cruz, Calif., USA, catalogue # Z-5), and secondaryHRP-conjugated rabbit anti-mouse IgG (RAMPO). The blots were developedusing Western Lightning Chemiluminescence Reagent Plus (PerkinElmer LifeSciences, MA, USA). Stable clones were grown in the presence of 250 μgml⁻¹ G-418. For pull-down experiments, conditioned medium with 5% FCS ofstable clones that produce constructs of interest was used. Forpurification purposes, cells of a stable clone of tPA F-EGF-GST weretransferred to triple-layered culture flasks and grown in medium with0.5% v/v Ultroser G (ITK Diagnostics, Uithoorn, The Netherlands). Mediumwas refreshed every three to four days. TPA F-EGF-GST was isolated fromthe medium on a Glutathione Sepharose 4B (Amersham Biosciences, Uppsala,Sweden) column and eluted with 100 mM reduced glutathione (RocheDiagnostics, Mannheim, Germany). Purity of the construct was checkedwith SDS-PAGE followed by Coomassie staining or Western blotting. Fromthese analyses, it is clear that some GST is present in the preparation.Purified tPA F-EGF-GST was dialyzed against PBS and stored at −20° C.

Example 26 Purification of GST-Tagged tPA-F-GST and RPTPμ-GST

Medium was concentrated twenty-fold using Nanosep 10K Ω centrifugaldevices (Pall Gelman Laboratory, MI, USA) and incubated with glutathionecoupled to Sepharose 4B, according to the manufacturer's recommendations(Pharmacia Biotech, Uppsala, Sweden). Bound constructs were washed withPBS and eluted with 10 mM of glutathione in 50 mM Tris-HCl pH 8.0.Constructs were stored at −20° C., before use.

Example 27 Amyloid Pull-Down

Conditioned medium of BHK cells expressing GST-tagged tPA F, F-EGF, EGF,K1, F-EGF-K1, FXII F, HGFa F, Fn F4, Fn F5, Fn F4-5 and GST was used foramyloid binding assays. At first, constructs were adjusted toapproximately equal concentration using Western blots. Qualitativebinding of the recombinant fragments are evaluated using a “pull-down”assay. To this end, the recombinantly made fragments are incubated witheither Aβ or IAPP fibrils. Since these peptides form insoluble fibers,unbound proteins can be easily removed from the fibers followingcentrifugation. The pellets containing the bound fragments aresubsequently washed several times. Bound fragments are solubilized inSDS-sample buffer and analyzed by PAGE, as well as unbound proteins inthe supernatant fraction and starting material. The gels are analyzedusing immunoblotting analysis with the anti-GST antibody Z-5.

Example 28 Amyloid ELISA with tPA F-EGF-GST

In order to define the affinity of the purified tPA F-EGF-GSTrecombinant protein, ELISAs were performed with immobilized amyloid(poly)peptides and non-amyloid control ΔmIAPP. Twenty-five μg ml⁻¹ ofAβ, FP13, IAPP or ΔmIAPP was immobilized on Exiqon peptide immobilizerplates. A concentration series of tPA F-EGF-GST in the presence ofexcess εACA was added to the wells and binding was assayed usinganti-GST antibody Z-5. As a control, GST (Sigma-Aldrich, St. Louis, Mo.,USA, catalogue # G-5663) was used at the same concentrations.

Example 29 Immunohistochemistry: Binding of tPA F-EGF to Human AD Brain

Paraffin brain sections of a human inflicted with AD was a kind gift ofProf. Slootweg (Dept. of Pathology, UMC Utrecht). Sections weredeparaffinized in a series of xylene-ethanol. Endogenous peroxidaseswere blocked with methanol/1.5% H₂O₂ for 15 minutes. After rinsing inH₂O, sections were incubated with undiluted formic acid for ten minutes,followed by incubation in PBS for five minutes. Sections were blocked in10% HPS in PBS for 15 minutes. Sections were exposed for two hours with7 nM of tPA F-EGF-GST or GST in PBS/0.3% BSA. After three wash stepswith PBS, sections were overlayed with 200 ng ml⁻¹ anti-GST antibodyZ-5, for one hour. After washing, ready-to-use goat anti-rabbitPowervision (Immunologic, Duiven, The Netherlands, catalogue #DPVR-55AP) was applied and incubated for one hour. After washing,sections were stained for ten minutes with 3,3′-diamino benzidine(Sigma-Aldrich, St Louis, Mo., USA, catalogue # D-5905), followed byhematoxylin staining for ten seconds. After washing with H₂O, sectionswere incubated with Congo red according to the manufacturer'srecommendations (Sigma Diagnostics, St Louis, Mo., USA). Sections werecleared in xylene and mounted with D.P.X. Mounting Medium (Nustain,Nottingham, U.K.). Analysis of sections was performed on a Leica DMIRBEfluorescence microscope (Rijswijk, The Netherlands). Fluorescence ofCongo red was analyzed using an excitation wavelength of 596 nm and anemission wavelength of 620 nm.

Example 30 ELISA: Binding of tPA-F-GST and RPTPμ-GST to Human Ab (1-40)and Glycated Albumin

Binding of tPA-F-GST and RPTPμ-GST to fibrous amyloids human Aβ (1-40)and albumin-g6p was assayed with an ELISA. In brief, human Aβ (1-40),albumin-g6p, or buffer only, were coated on a peptide I Immobilizer or aprotein I Immobilizer, respectively. Wells were incubated with thepurified GST-tagged constructs or control medium and binding wasdetected using primary anti-GST monoclonal antibody 2F3 and RAMPO. Thewells were also incubated with 500 mM of tPA in the presence of 10 mM ofεACA. Binding of tPA is independent of the lysyl binding site located atthe kringle 2 domain. Binding of tPA was measured using primary antibody374B and RAMPO. Experiments were performed in triplicate and blankreadings of non-coated wells were substracted.

Example 31 Anti-AGE Antibodies

Antibodies against glucose-6-phosphate glycated bovine serum albuminwere elicited in rabbits using standard immunization schemes. Anti-AGE1was obtained after immunization with two-week glycated albumin-AGE(Prof. Dr. Ph.G. de Groot/Dr. I. Bobbink; unpublished data). Theantibody was purified from serum using a Protein G column. Anti-AGE2 wasdeveloped by Davids Biotechnologie (Regensburg, Germany). Afterimmunization with albumin-AGE:23, antibodies were affinity purified onhuman Aβ (1-40) conjugated to EMD Epoxy-activated beads (Merck,Darmstadt, Germany). Polyclonal mouse anti-AGE antibody was obtainedafter immunization with albumin-AGE:23 and human Aβ (1-40) in a molarratio of 9:1. Polyclonal serum was obtained using standard immunizationprocedures, which were performed by the Academic Biomedical ClusterHybridoma Facility (Utrecht University, The Netherlands). Subsequently,monoclonal antibodies were generated using standard procedures.

Example 32 ELISA: Binding of Antibodies Against Amyloid Peptides orGlycated Protein to Protein-AGE and Amyloid Fibrils

For ELISAs, amyloid compounds were immobilized on Exiqon peptide orprotein Immobilizers (Vedbaek, Denmark), as described before. Anti-AGEantibodies and commercially available anti-Aβ (1-42) H-43 (Santa CruzBiotechnology, Santa Cruz, Calif., USA) were diluted in PBS with 0.1%v/v Tween 20. Rabbit anti-human vitronectin K9234 was a kind gift of Dr.H. de Boer (UMC Utrecht) and was used as a negative control. For ELISAswith mouse polyclonal anti-albumin-AGE/Aβ, control serum with antibodyelicited against an unrelated protein was used. Binding of mousepolyclonal anti-albumin-AGE/Aβ was performed using a dilution series ofserum in PBS/0.1% Tween 20. For competitive binding assays with IAPP,anti-AGE1 was pre-incubated with varying IAPP concentrations. The IAPPfibrils were spun down and the supernatant was applied in triplicate towells of an ELISA plate coated with Aβ. Competitive binding assays withmultiligand cross-β structure binding serine protease tPA were performedin a slightly different way. Coated Aβ and IAPP are overlayed with ananti-AGE1 or anti-Aβ (1-42) H-43 concentration related to the k_(D),together with a concentration series of tPA. A 10⁷-10⁴ times molarexcess of lysine analogue εACA (10 mM) was present in the binding bufferin order to avoid binding of tPA to lysine residues of Aβ and IAPP,which would be independent of the presence of amyloid structures.

Example 33 Pull-Down Assay with Amyloid Peptides and Rabbit Anti-AGE1Antibody

Anti-AGE1 was incubated with amyloid aggregates of Aβ (16-22), Aβ (1-40)and IAPP. After centrifugation, pellets were washed three times withPBS/0.1% Tween 20, dissolved in non-reducing sample buffer (1.5% (mn/v)sodium dodecyl sulphate, 5% (v/v) glycerol, 0.01% (m/v) bromophenolblue, 30 mM Tris-HCl pH 6.8). Supernatant after pelleting of the amyloidfibrils was diluted 1:1 with 2× sample buffer. Samples were applied to apolyacrylamide gel and after Western blotting, anti-AGE1 was detectedwith SWARPO.

Example 34 Immunohistochemical Analysis of the Binding of Anti-AGE2 toan Amyloid Plaque in a Section of a Human Brain Inflicted by AD

Rabbit anti-AGE2, affinity purified on an Aβ column, was used forassaying binding properties towards amyloid plaques in brain sections ofa human with AD. The procedure was performed essentially as describedabove. To avoid eventual binding of 11 μg ml⁻¹ anti-AGE2 to protein-AGEadducts or to human albumin in the brain section, 300 nM of g6p-glycateddipeptide Gly-Lys was added to the binding buffer, together with 0.3%m/v BSA. After the immunohistochemical stain, the section was stainedwith Congo red.

Example 35 Sandwich ELISA for Detection of Amyloid Albumin-AGE inSolution

For detection of amyloid cross-β structure in solutions, the sandwichELISA approach was used. Actilyse tPA was immobilized at a concentrationof 10 μg ml⁻¹ to wells of a 96-wells protein Immobilizer plate (Exiqon,Vedbaek, Denmark). Concentration series of albumin-AGE:23 andalbumin-control:23 were added to the tPA-coated wells, as well as tonon-coated control wells. Binding of amyloid structures was detectedupon incubation with 1 μg ml⁻¹ anti-Aβ (1-42) H-43 (Santa CruzBiotechnology, Santa Cruz, Calif., USA) and subsequently 0.3 μg ml⁻¹SWARPO, followed by ortho-phenylene-diamine/H₂O₂/H₂SO₄ stain.

Example 36 Cross-β Structure is Present in Fibrin and in SyntheticPeptides Derived from Fibrin

It has been demonstrated herein that a fibrin clot stains with Congo red(not shown) and exhibits Thioflavin T fluorescence (FIG. 2, Panel A),indicative of the presence of amyloid structure in a fibrin clot. UsingCongo red staining (not shown), circular dichroism measurements andX-ray diffraction analysis, it is shown that synthetic peptides derivedfrom the sequence of fibrin adopt cross-β structure (FIG. 2, Panels Band C). These peptides were previously found to possess tPA-binding andtPA-activating properties.¹⁸ The presence of cross-β structure in thesepeptides was found to correlate with the ability to stimulatetPA-mediated plasminogen activation (FIG. 2, Panel D).

These data provide evidence for physiological occurrence/relevance forformation of cross-β structure and the role of this structural elementin binding of tPA to fibrin.

Example 37 Aβ Contains Cross-β Structure, Binds Plasmin(ogen) and tPA,Stimulates Plasminogen Activation, Induces Matrix Degradation andInduces Cell Detachment that is Aggravated by Plasminogen and Inhibitedby CpB

To test whether tPA, plasminogen and plasmin bind Aβ, solid-phasebinding assays were performed. Aβ was coated onto plastic 96-well platesand binding of the peptide to either plasmin(ogen) or to tPA wasassessed by overlaying the coated peptide with increasing concentrationsof either tPA, plasminogen or plasmin. Binding was assessed usingspecific antibodies to either plasmin(ogen) or to tPA by performingELISA. FIG. 3, Panel A, shows that tPA binds to Aβ with a k_(D) of about7 nM, similar to the k_(D) of tPA binding to fibrin.⁵⁵ In contrast, Nodetectable binding of plasminogen to Aβ was found (FIG. 3, Panel B).However, activated plasminogen (plasmin) does bind to Aβ and does sowith a k_(D) of 47 nM. The fact that (active) plasmin, but not(inactive) plasminogen, binds to Aβ suggests that plasmin activity and,hence, the generation of free lysines, is important for binding ofplasmin to Aβ. To test this, use was made of the lysine analogueε-aminocaproic acid (εACA) and binding of plasmin and tPA to Aβ wastested in its presence. It is shown herein that the binding of plasminto Aβ is completely abolished in the presence of εACA (FIG. 3, Panel D).In contrast, εACA has no effect on the tPA-Aβ interaction (FIG. 3, PanelC). Thus, it was concluded that plasmin binds to free lysines that weregenerated during the incubation period, presumably via itslysine-binding Kringle domain(s). In line with this, the k_(D) ofplasminogen Kringle domain binding to free lysines in fibrin is similarto the k_(D) for plasmin binding to Aβ.

The kinetics of plasminogen activation in the absence and the presenceof Aβ was investigated. As has been published before by Kingston etal.,²⁴ it was found that Aβ potently stimulates the activation ofplasminogen by tPA (FIG. 4, Panel A). However, it was found that thereaction proceeds with second-order, rather than with first-order,kinetics. The possibility was considered that the generation of freelysines during the reaction was causing this phenomenon (see below).tPA-mediated plasmin generation has been implicated in neuronal celldeath caused by ischemia or by excitotoxic amino acids. Recent datasuggest that plasmin can degrade Aβ and thereby prevents Aβtoxicity.^(56, 57) It was found that 48 hours following the addition ofAβ to a culture of differentiated N1E-115 cells, the majority of cellshad died and detached from the matrix (not shown). When added togetherwith Aβ, plasmin (up to 100 mM) was unable to ameliorate Aβ-induced celldetachment. Even prolonged pre-incubations of Aβ with 100 nM plasmin didnot affect Aβ-induced cell detachment (FIG. 4, Panel B). Subsequently,the possibility was considered that plasmin generation may potentiaterather than inhibit Aβ-induced cell detachment and survival. To testthis, N1E-115 cells were exposed to suboptimal concentrations of Aβ andlow concentrations of plasminogen for 24 hours. In the absence of Aβ,plasminogen has no effect on cell adhesion (FIG. 4, Panel C). However,plasminogen has a dramatic potentiating effect on Aβ-induced celldetachment. The minimal levels of plasminogen that are required topotentiate Aβ-induced cell detachment (10-20 μg/ml) are well below thosefound in human plasma (250 μg/ml). Plasmin-mediated degradation of theextracellular matrix molecule laminin precedes neuronal detachment andcell death in ischemic brain. Whether Aβ-stimulated plasmin generationleads to laminin degradation was tested. Cell detachment was accompaniedby degradation of the extracellular matrix protein laminin (FIG. 4,Panel D).

The possibility was considered that the generation of free lysinesduring Aβ-stimulated plasmin formation was responsible for the observedsecond-order kinetics. To test this, use was made of carboxypeptidase B(CpB), an enzyme that cleaves of C-terminal lysine and arginineresidues, and the CpB-inhibitor CPI. FIG. 5, Panel A, shows that in thepresence of CpB, the generation of plasmin is greatly diminished.Furthermore, this effect depends on CpB activity as it is abolished byco-incubation with CPI. FIG. 5, Panel A, also shows that CpB does notcompletely abolish Aβ-stimulated plasmin generation, but that thereaction proceeds with slow first-order kinetics. These data suggestthat the (plasmin-mediated) generation of free lysines during thereaction is essential for efficient Aβ-stimulated plasmin generation,presumably by supporting plasminogen and tPA binding through interactionwith their respective Kringle domains. A similar dependency on thegeneration of C-terminal lysines has been shown for efficientfibrin-mediated plasmin generation.⁵⁸ These results show thatAβ-stimulated plasmin formation is regulated by carboxypeptidase B invitro. Thus, if cell detachment is the result of plasmin generation, CpBmay affect Aβ-induced cell detachment and/or viability. It is shownherein that cell detachment induced by plasminogen and Aβ is completelyprevented by co-incubation with CpB (FIG. 5, Panels B and C). This isaccompanied by a complete inhibition of Aβ-stimulated plasmin formation,both in the medium and on the cells (FIG. 5, Panel D).

Example 38 Endostatin can Form Amyloid Fibrils Comprising Cross-βStructure

Using Congo red staining (not shown), X-ray diffraction analysis andTEM, the presence of cross-β structure in aggregated endostatin fromEscherichia coli, as well as from Pichia pastoris, and the ability ofendostatin to form amyloid fibrils (FIG. 6, Panels A and B) has beendemonstrated. It was found that bacterial endostatin produced reflectionlines at 4.7 Å (hydrogen-bond distance), as well as at 10-11 Å(inter-sheet distance). The reflection lines show maximal intensities atopposite diffraction angles. The fiber axis, with its 4.7 Å hydrogenbond repeat distance, is oriented along the vertical capillary axis.This implies that inter-sheet distance of 10-11 Å is perpendicular tothese hydrogen bonds. This is consistent with the protein being across-β sheet conformation with a cross-β structure. Intramolecular βsheets in a globular protein cannot cause a diffraction pattern that isordered in this way. From the amount of background scattering, itfollows that only part of the protein takes part in cross-β structureformation. It was found that the presence of cross-β structures inendostatin correlates with its ability to stimulate tPA-mediatedplasminogen activation (FIG. 6, Panel C) and correlates with neuronalcell death (FIG. 6, Panel D).

Herein, it is demonstrated that endostatin is an example of a denaturedprotein that is able to stimulate the suggested cross-β pathway.

Example 39 IAPP Binds tPA and Stimulates tPA-Mediated PlasminogenActivation

Amyloid deposits of IAPP are formed in the pancreas of type II diabeticpatients.⁵⁹ IAPP can cause cell death in vitro and is, therefore,thought to contribute to destruction of β-cells that is seen in vivo,which leads to insufficient insulin production. IAPP forms fibrilscomprising cross-β structure.⁶⁰

Whether IAPP could stimulate tPA-mediated plasminogen activation wastested (FIG. 7). Indeed, similar to Aβ, IAPP can enhance the formationof plasmin by tPA.

Example 40 Glycated Albumin Binds Thioflavin T and tPA, and Aggregatesas Amyloid Fibrils Comprising Cross-β Structure

It has been demonstrated that glycation of several proteins can induceor increase the ability of these proteins to bind tPA and stimulatetPA-mediated plasmin formation.^(22,61) It is shown herein thatglycation of albumin with g6p not only confers high-affinity tPA bindingto albumin (FIG. 8, Panel A), but also leads to its ability to bindThioflavin T (FIG. 8, Panel C). Binding of tPA can be competed withCongo red (FIG. 8, Panel B). In addition, binding of Thioflavin T toglycated albumin can be competed by tPA (FIG. 8, Panels D and E). Thefact that Congo red and/or Thioflavin T and tPA compete illustrates thatthey have either the same or overlapping binding sites.

Analyses with TEM of the g6p-modified albumin preparations revealed thatafter a four-week incubation, amorphous albumin aggregates are formed(FIG. 8, Panel G), which exhibits a CD spectrum indicative for thepresence of 7% of the albumin amino-acid residues in β-sheet (Table I).Prolonged incubation up to 23 weeks resulted in a switch to highlyordered sheet-like fibrous structures, with a length of approximately500 nm and a diameter ranging from about 50 to 100 nm (FIG. 8, Panel H).These fibers showed an increase to 19% β-sheet when analyzed with CDspectropolarimetry (Table I). Albumin from a different batch that wasglycated in the same way already showed bundles of fibrous aggregatesafter a two-week period of incubation (FIG. 8, Panel I), whereas anincrease in β-sheet content is not detected with CD spectropolarimetry(Table I). In each bundle, about ten separate linear 3-5-nm-wide fiberswith a length of 200-300 nm can be identified. On top of each bundle,regularly distributed spots are seen with a diameter of approximately 5nm. These spots may be accounted for by globular albumin molecules thatare bound to the fibers or, alternatively, that are partly incorporatedin the fibers. Aggregates were absent in control albumin (not shown) andno β-sheets were measured using CD spectropolarimetry (Table I). Thefibrous structures obtained after two-week and 23-week periods ofglycation enhance the fluorescence of Thioflavin T (ThT) in a similarway, whereas the amorphous precipitates obtained after four weeks hardlyincreased the fluorescent signal.

X-ray fiber diffraction analyses revealed that albumin-g6p (23 weeks)comprises a significant amount of crystalline fibers (FIG. 8, Panels Jand L), whereas diffraction patterns of albumin-g6p (2 weeks) andalbumin-g6p (4 weeks) show features originating from amorphousprecipitated globular protein very similar to the patterns obtained foralbumin controls (FIG. 8, Panel K). For albumin-g6p (23 weeks), the 4.7Å repeat corresponds to the characteristic hydrogen-bond distancebetween β-strands in β-sheets. The 2.3 and 3.3 Å repeats have apreferred orientation perpendicular to the 4.7 Å repeat (FIG. 8, PanelM). Combining the 2.3 and 3.3 Å repeats suggests that the fiber axis isoriented perpendicular to the direction of the hydrogen bonds, with arepeat of 6.8 Å. This dimension corresponds to the length of two peptidebonds and indicates that β-strands run parallel to the fiber axis. Thisimplies that the albumin-g6p (23 weeks) structure is composed of cross-βstructure consisting of packed β-sheets of hydrogen-bonded chains (FIG.8, Panel N). A similar orientation is found in amyloid fibrils of thefirst predicted a-helical region of PrP^(c).⁶² When the a-axis is 9.4 Å,or alternatively 4.7 Å, and the c-axis is 6.8 Å, the 2.5 and 6.0 Årepeats can only be indexed as (h k l). This implies a highly orderedb-axis repeat corresponding to the inter β-sheet distance. With a-axisand c-axis of 4.7 or 9.4 Å and 6.8 Å, respectively, the strong 3.8 Årepeat should be indexed as (2 0 1) or (1 0 1). Considering allobservations, it is clear that the albumin-g6p fibers (23 weeks) arebuilt up by cross-β structures, a characteristic feature of amyloidfibrils.

These results show that due to incubation and/or modification with sugarmoieties, cross-β structures in albumin are formed that are able tosupport tPA binding.

Example 41 Glycation of Hemoglobin Induces tPA Binding and FibrilFormation

Incubation of human hemoglobin with g6p resulted in high-affinity tPAbinding (FIG. 9, Panel A). Amorphous aggregated Hb-g6p adducts includingfibrils were observed with TEM (FIG. 9, Panel B), whereas control Hb didnot aggregate (not shown). Human Hb of diabetes mellitus patients hasthe tendency to form fibrillar aggregates once more than 12.4% of the Hbis glycated (Table II).

Example 42 Amyloid Albumin is Formed Irrespective of the OriginalCarbohydrate (Derivative)

From the above-listed observations, it is clear that modification of—NH₂ groups of albumin with g6p induces formation of amyloid cross-βstructure. The next question addressed was whether triggering ofrefolding of globular albumin into an amyloid fold was a restrictedproperty of g6p, or whether amyloid formation occurs irrespective of theoriginal carbohydrate or carbohydrate derivative used for AGE formation.Albumin solutions were incubated for 86 weeks at 37° C. with 1 M g6p,250 mM DL-glyceraldehyde/100 mM NaCNBH₃, 1 M P-D-(−)-fructose, 1 MD(+)-glucose, 500 mM glyoxylic acid/100 mM NaCNBH₃, and correspondingPBS and PBS/NaCNBH₃ buffer controls. Glyceraldehyde and glyoxylic acidare carbohydrate derivatives that are precursors of AGE in Maillardreactions.^(63,64) After 86 weeks, albumin-glyceraldehyde andalbumin-fructose were light-yellow/brown suspensions. Controls werecolorless and clear solutions. Albumin-glucose and albumin-glyoxylicacid were clear light-yellow to light-brown solutions. Albumin-g6p:86was a clear and dark brown solution. AGE formation was confirmed byautofluorescence measurements using AGE-specific excitation/emissionwavelengths (not shown), binding of moab anti-AGE 4B5 (not shown) andbinding of poab anti-AGE (not shown). As expected, albumin-glyoxylicacid did not show an autofluorescent signal due to the fact that(mainly) non-fluorescent carboxymethyl-lysine (CML) adducts areformed.⁶³

The autofluorescence data and the binding of AGE-specific antibodieslisted above show that various carbohydrates and carbohydratederivatives can lead to similar AGE structures. Using g6p as thestarting point for AGE formation, it is shown that albumin adoptedamyloid properties similar to those observed in well-studied fibrils ofAβ and IAPP. Therefore, the presence of amyloid structures in thealbumin-AGE adducts obtained with alternative carbohydrates andderivatives was tested for. Fluorescence of albumin-AGE—ThT solutions(FIG. 10, Panel J) and of air-dried albumin-AGE preparations that wereincubated with Congo red (FIG. 10, Panels A-I) was measured. Incubationof albumin with glyceraldehyde, glucose or fructose resulted in anincreased fluorescent signal of ThT (FIG. 10, Panel J). Aftersubtraction of background signals of ThT and buffer, no specificamyloid—ThT fluorescence was measured for albumin-glyoxylic acid andbuffer controls. Albumin-g6p, albumin-glyceraldehyde andalbumin-fructose gave a Congo red fluorescent signal similar to signalsof Aβ and IAPP (FIG. 10, Panels C-E, G and H). With albumin-glucose, auniformly distributed pattern of fluorescent precipitates is observed(FIG. 10, Panel F). With albumin-glyoxylic acid and buffer controls,hardly any staining is observed (FIG. 10, Panels A, B and I). These ThTand Congo red fluorescence data show that, in addition to albumin-g6p,albumin-glyceraldehyde, albumin-glucose and albumin-fructose haveamyloid-like properties. To further substantiate these findings, bindingof amyloid-specific serine protease tPA in an ELISA was tested for. Theenzyme bound specifically to albumin-g6p, albumin-glyceraldehyde,albumin-glucose and albumin-fructose (FIG. 10, Panels K and L) and topositive controls Aβ and IAPP, as was shown before.⁴⁹ No tPA binding isobserved for albumin-glyoxylic acid and buffer controls.

From the ThT, Congo red and tPA data, it is clear that inducing amyloidproperties in albumin is not an exclusive property of g6p. Apparently, aspectrum of carbohydrates and carbohydrate derivatives comprising g6p,glucose, fructose, glyceraldehyde, and likely more, has the capacity totrigger the switch from a globular native fold to the amyloid cross-βstructure fold upon their covalent binding to albumin.

Example 43 Analysis of Congo Red Binding and tPA Binding to Aβ

It has been demonstrated that Aβ can bind tPA and Congo red. It is shownthat the binding of tPA to Aβ can be competed by Congo red (FIG. 11).These results support the findings herein that structures in Aβ, fibrinand glycated albumin are similar and are able to mediate the binding totPA.

Example 44 Binding of human FXII to amyloid peptides and proteins thatcontain the cross-β structure fold.

The graphs in FIG. 12 show that FXII binds specifically to all amyloidcompounds tested. k_(D)s for hAβ (1-40), FP13, albumin-AGE and Hb-AGEare approximately 2, 11, 8 and 0.5 nM, respectively. The data obtainedwith the competitive FXII—tPA ELISA show that tPA efficiently inhibitsbinding of FXII to amyloid (poly)peptides (FIG. 12). From these data, itis concluded that FXII and f.l. tPA compete for overlapping bindingsites on hAβ (1-40). K2P-tPA does not inhibit FXII binding. Binding ofFXII to albumin-AGE is also effectively abolished by tPA but not byK2P-tPA, similar to what was observed for hAβ (1-40). This indicatesthat FXII may bind in a similar manner to hAβ (1-40) and albumin-AGE. Inaddition, these data show that the first three domains of tPA (finger,EGF-like, kringle 1) seem to be involved in the inhibitory effect off.l. tPA on interactions between FXII and amyloid hAβ (1-40) oralbumin-AGE. Using a dot-blot assay, binding of FXII to spotted amyloidhΔIAPP and hAβ (1-40) was tested. No binding of FXII was observed fornegative controls PBS and mΔIAPP (FIG. 12). However, FXII specificallybound to hAβ (1-40), in agreement with an earlier report,⁶⁵ as well asto hΔIAPP (FIG. 12). These data, together with the ELISA data shown inFIG. 12, Panels A-F, suggest that FXII can bind to polypeptides that donot share amino acid sequence homology, though which share the cross-βstructure fold. This is in accordance with the recent data oninteractions between tPA and polypeptides that contain theamyloid-specific fold.

Example 45 Binding of tPA to the Cross-β Structure-Containing Molecules,Aβ and Glycated Albumin Requires the Presence of an N-Terminal Region intPA, Which Contains the Finger Domain

Several domains in tPA have been shown to mediate binding to fibrin orfibrin fragments.^(12,53,66,67) However, it is unknown which domain oftPA is needed for binding to Aβ or other cross-β structure-containingmolecules. It is shown that a mutated tPA, termed reteplase, which lacksthe N-terminal finger, EGF and kringle 1 domain (K2-tPA), is unable tobind cross-β structure-comprising molecules (FIG. 13, Panels A and B).These results suggest that the N-terminal region is required for bindingof tPA to fibrils comprising cross-β structure.

Example 46 Expression and Purification of tPA-F-GST and RPTP-GST

Purification of the GST-tagged constructs tPA-F-GST andRPTPμ-GST(control) from 293T medium using glutathione coupled toSepharose 4B beads resulted in single bands of approximately 35 kDa and150 kDa, respectively (not shown). Traces of BSA, originating from theFCS used in the medium, were also present.

Example 47 ELISA: Binding of tPA-F-GST and RPTP-GST to human Aβ (1-40)and Glycated Albumin

In the ELISA, control tPA bound to both human Aβ (1-40) and albumin-g6pin the presence of excess εACA (FIG. 13, Panel C). This shows that inthe assay used, tPA is capable of binding to fibrous amyloids in akringle 2-independent manner. The tPA-F domain bound to human Aβ (1-40)and to albumin-g6p, whereas no binding was observed with RPTPμ-GST.Therefore, binding observed with tPA-F-GST is specific and does notoriginate from the GST tag. This result points to the tPA finger domainas a specific domain designed by nature for binding tocross-β-structured amyloid fibrils.

cDNA constructs in pcDNA3 of the F, F-EGF, EGF, F-EGF-K1 and K1fragments of human tPA was prepared. Recombinant proteins with aC-terminal GST tag were expressed in BHK cells and secreted to themedium. Medium from BHK cells expressing the GST tag alone was used as acontrol. Conditioned medium was used for pull-down assays using Aβ andIAPP fibrils, followed by Western blot analyses. Efficient binding to Aβis evident for all three tPA mutants that contain the finger domain,i.e., F-GST, F-EGF-GST and F-EGF-K1-GST (FIG. 13, Panel D). The K1-GSTand EGF-GST constructs, as well as the GST tag alone, remain in thesupernatant after Aβ incubation. A similar pattern was obtained afterIAPP pull-downs (not shown).

Binding of purified tPA F-EGF-GST, recombinant f.l. Actilyse tPA and aGST control to immobilized amyloid Aβ, amyloid fibrin fragment α₁₄₈₋₁₆₀FP13, amyloid IAPP and to non-amyloid mΔIAPP control was compared (FIG.13, Panels E-G). Full-length tPA and tPA F-EGF-GST bind to all threeamyloid peptides; for Aβ k_(D)s for tPA and F-EGF are 2 and 2 nM,respectively; for FP13, 5 and 2 nM; for IAPP, 2 and 13 nM. No binding tonon-amyloid mΔIAPP is observed (FIG. 13, Panel E). GST does not bind toFP13 and IAPP, while some binding is detected to Aβ. This may reflectthe small fraction of GST that bound to Aβ in the pull-down assay (FIG.13, Panel D).

With immunohistochemical analysis, binding of the purified recombinanttPA F-EGF-GST construct to paraffin sections of human brain inflicted byAD was tested. Presence of amyloid depositions was confirmed by theDept. of Pathology (UMC Utrecht) using standard techniques. In theexperiments, these amyloid depositions were located using Congo redfluorescence (FIG. 13, Panels I, K and M). In FIG. 13, Panels H-K, it isclearly seen that areas that are positive for Congo red bindingcoincides with areas that are positive for tPA F-EGF-GST binding.Control stain with GST does not show specific binding of the tag alone(FIG. 13, Panels L and M).

At present, based on sequential and structural homology, next to tPA,three proteins are known that contain one or more finger domains, i.e.,HGFa (one F domain), FXII (one F domain), Fn (one stretch of six Fdomains, two stretches of three F domains). From the above-listedresults, it was concluded that the F domain of tPA (SEQ ID NO: 3) playsa crucial role in binding of tPA to amyloid (poly)peptides. It washypothesized that the finger domain could be a general cross-βstructure-binding module. Presently, four proteins, tPA, FXII, HGFa andfibronectin, are known that contain a finger motif. FIG. 14, Panel A,schematically depicts the localization of the finger module in therespective proteins. FIG. 14, Panel B, shows an alignment of the humanamino acid sequences of the finger domains in these four proteins. (SEQID NOs: 3-17) FIG. 14, Panel C, shows a schematic representation of thethree-dimensional structure of the finger domain of tPA (SEQ ID NO: 3),and of the fourth and fifth finger domain of fibronectin (SEQ ID NOs: 9and 10). To test the hypothesis that finger domains in general bindamyloid, the F domains of HGFa (SEQ ID NO: 5) and FXII (SEQ ID NO: 4),as well as the fourth and fifth F domain of Fn (SEQ ID NOs: 9 and 10),which are known for their capacity to bind to fibrin,⁶⁸ were cloned.Using a pull-down assay, it was shown that Fn F4-GST and Fn F4-5-GST, aswell as FXII F-GST and HGFa F-GST, specifically bind to Aβ (FIG. 13,Panels M and N) and IAPP (not shown). Fn F5-GST binds to Aβ to someextent, however, it is extracted less efficiently from the medium andseems to be party released during the washing procedure of the amyloidpellet (FIG. 13, Panel M). No construct was left in the medium afterextraction of positive control tPA F-EGF-GST, whereas no negativecontrol GST was detected in the pellet fraction (not shown). These datashow that binding to amyloid (poly)peptides is not a unique capacity ofthe tPA F domain (SEQ ID NO: 3), yet a more general property of the Fdomains tested. Moreover, these data indicate that observed binding ofFXII to amyloid (poly)peptides, as shown in FIG. 13, Panels A and H, andby Shibayama et al.,⁶⁵ is regulated via the F domain.

Example 48 Amyloid-Binding Domain of tPA

The finger domain of tPA has been shown to be of importance forhigh-affinity binding to fibrin.^(12,66) The present results usingRETEPLASE® (K2-P tPA) and F-tPA, F-EGF-tPA and F-EGF-K1-tPA, indicate animportant role for the N-terminal finger domain of tPA in binding tostimulatory factors other than fibrin. Thus far, all of these factorsbind Congo red and contain cross-β structure. Furthermore, the bindingsite of fibronectin for fibrin has been mapped to the finger domaintandem F4-F5.⁶⁸ It has been demonstrated that plasminogen activation byfull-length tPA, in the presence of fibrin fragment FCB2, can beinhibited by fibronectin.⁶⁹ Taken together, these observations suggestthat tPA and fibronectin compete, via their finger domain, for the sameor overlapping binding sites on fibrin. The data now shows that the F4-5domains of Fn (SEQ ID NOs: 9 and 10) bind to amyloid Aβ.

Example 49 Binding of Anti-AGE Antibodies to Amyloid (Poly)Peptides andBinding of Anti-Aβ to Protein-AGE Adducts

Recently, O'Nuallain and Wetzel⁷⁰ showed that antibodies elicitedagainst a peptide with amyloid characteristics can bind to any otherpeptide with similar amyloid properties, irrespective of amino acidsequence. Based on these data and on the observations that tissue-typeplasminogen activator and factor XII can bind to a family ofsequence-unrelated polypeptides that share the amyloid-specific cross-βstructure fold, a broader class of proteins can display affinity towardsthis structural unit, rather than towards a linear or conformationalepitope, built up by specific amino acid residues. This prompted thequestion as to whether antibodies elicited against albumin-AGE, thatcontain the amyloid cross-β structure fold, also display the broad-rangespecificity towards any (poly)peptide which bears this cross-β structurefold.

In an ELISA set-up, α-AGE1, which was elicited against g6p-glycatedalbumin-AGE, binds to amyloid albumin-AGE:23 (K_(d)=66 nM) and Hb-AGE:32(K_(d)=20 nM), as well as to Aβ (1-40) (K_(d)=481 nM) and IAPP (K_(d)=18nM) (FIG. 15, Panels A-C). Negative controls were non-glycated albuminand Hb, non-amyloid peptide mouse ΔIAPP for IAPP and polyclonalanti-human vitronectin antibody α-hVn K9234 for Aβ. To test whether thesame fraction of α-AGE1 binds to IAPP and Aβ, the antibody waspre-incubated with IAPP fibrils, followed by pelleting of the fibrils,together with the possible amyloid-binding fraction of α-AGE1. Bindingof α-AGE1, left in the supernatant, to Aβ (1-40) was reduced (FIG. 15,Panel D). This indicates that the same fraction of α-AGE1 binds to IAPPand Aβ (1-40). With a pull-down assay, the binding of anti-AGE1 toamyloid peptides in an alternative way was assessed. After incubation ofanti-AGE1 solutions with amyloid fibrils Aβ (16-22) (FIG. 15, Panel E,lanes 1-2), Aβ (1-40) (FIG. 15, Panel E, lanes 4-5), and IAPP (FIG. 15,Panel E; lanes 6-7), and subsequent pelleting of the amyloid fibrils,the supernatant was completely depleted from α-AGE1 by Aβ (16-22). WithIAPP, approximately 50% of the antibody is found in the amyloidfraction, whereas less antibody is pelleted with Aβ (1-40). These dataobtained in a complementary way, again show that anti-AGE 1 can bind toamyloid peptides, which share no amino-acid sequence homology withalbumin-AGE:23, though which share the cross-β structure fold. Inaddition, the observation that binding of tPA to amyloid peptidesinhibits binding of anti-AGE1, also indicates that anti-AGE1, like tPA,binds to the cross-β structure fold (FIG. 15, Panels F and G). Theobservation that tPA reduces anti-AGE1 binding to Aβ to a lesser extentthan the reduction seen with IAPP, is putatively related to the highernumber of anti-AGE1-binding sites on coated Aβ, when compared with IAPP(see FIG. 15, Panels B and C), and to the higher affinity of tPA forIAPP (k_(D)=6 nM) than for Aβ (k_(D)=46 nM), when using Exiqon ELISAplates (not shown). The binding data together suggest that anti-AGE1binds to this amyloid fold, irrespective of the (poly)peptide that bearsthe cross-β structure fold, which identifies anti-AGE1 as a member ofthe class of multiligand cross-β structure-binding proteins.

Based on the above-listed results obtained with anti-AGE1, testing wasdone as to whether commercially available rabbit anti-human Aβ (1-42)H-43 also displays broad-range specificity towards any (poly)peptidewith unrelated amino acid sequence, although with amyloidcharacteristics. Indeed, with an ELISA, it was shown that H-43 not onlybinds to Aβ (1-40), but also to IAPP and albumin-AGE (FIG. 15, Panel H).In addition, binding of H-43 to immobilized IAPP was effectivelydiminished by tPA (FIG. 15, Panel I). These observations together showthat anti-Aβ (1-42) H-43 can bind to other amyloid (poly)peptides in away similar to multiligand cross-β structure-binding protein tPA.

ELISAs with polyclonal mouse anti-albumin-AGE/Aβ show that the antibodynot only binds to these antigens, but that it specifically binds toother amyloid peptides than those used for immunization (FIG. 15, PanelsJ-L). Similar to the rabbit anti-AGE1 antibody and anti-Aβ (1-42) H-43,anti-albumin-AGE/Aβ displays affinity for the amyloid peptides tested,irrespective of amino acid sequence. This suggests that also mouseanti-albumin-AGE/Aβ is a multiligand amyloid-binding antibody.

Based on the amyloid-binding characteristics of anti-AGE1, anti-Aβ(1-42) H-43 and anti-albumin-AGE/Aβ, the amyloid-binding fraction ofanti-AGE2, which is elicited against albumin-AGE:23, with Aβ fibrilsirreversibly coupled to a column, was purified. This fraction was usedfor immunohistochemical analysis of a human brain section that isinflicted by Alzheimer's disease. In FIG. 15, Panel M, it is clearlyseen that the antibody binds specifically to the spherical amyloiddeposition, indicated by the Congo red fluorescence shown in FIG. 15,Panel N.

The finding that anti-amyloid and anti-AGE antibodies display affinityfor a broad range of sequentially unrelated (poly)peptides as long asthe cross-β structure fold is present, is in agreement with the recentlypublished data by O'Nuallain and Wetzel⁷⁰ and Kayed et al.⁷¹ Fromseveral older reports in literature, it can be distilled thatanti-cross-β antibodies can be obtained. For example, cross-reactiveantibodies against fibrin and Aβ and against Aβ and hemoglobin aredescribed.^(72,73) It is indicated herein that fibrinogen andhemoglobin-AGE adopt the cross-β structure fold, which suggests that thecross-reactivity observed for anti-Aβ antibodies was, in fact, bindingof anti-cross-β structure antibodies to similar structural epitopes onAβ, fibrinogen and hemoglobin.

Based on the results with the poly-clonal anti-AGE and amyloidantibodies, it was hypothesized that anti-cross-β structure antibodiescould be obtained. Therefore, the spleen of mice immunized with glycatedBSA and Aβ was fused with myeloma cells. Subsequently, potentialanti-cross-β structure antibodies were selected by examining binding ofhybridoma-produced antibodies to glycated hemoglobin and IAPP. Usingthis procedure, a monoclonal antibody 3H7 was isolated that recognizesglycated hemoglobin, as well as several peptides that contain thecross-β structure (FIG. 16). No binding was observed to unglycatedhemoglobin or a synthetic peptide that does not form amyloid fibrils(mΔIAPP).

Example 50 Sandwich ELISA: Fishing Amyloid Structures from Solution

Using a sandwich ELISA approach with coated tPA that was overlayed withamyloid albumin-AGE:23 in solution, followed by detection withbroad-range anti-Aβ (1-42) H-43 (FIG. 17), cross-β structure-containingproteins in solution were detected.

It is herein disclosed that the three-dimensional structures of the tPAfinger domain^(74,75) and the fibronectin finger domains 4-5^(75,76)reveal striking structural homology with respect to local charge-densitydistribution. Both structures contain a similar solvent-exposed stretchof five amino acid residues with alternating charge; for tPA, Arg7,Glu9, Arg23, Glu32, Arg30; and for fibronectin, Arg83, Glu85, Lys87,Glu89, Arg90, located at the fifth finger domain, respectively. Thecharged-residue alignments are located at the same side of the fingermodule. These alignments may be essential for fibrin binding.

Based on the observations, results and the herein-disclosedsimilarities, it is shown that the same binding sites for tPA becomepresent in all proteins that bind and activate tPA and that this bindingsite comprises a cross-β structure.

Taken together, the data shows that a cross-β structure is aphysiological relevant quaternary structure element in which appearanceis tightly regulated and in which occurrence induces a normalphysiological response, i.e., the removal of unwanted biomolecules. Tothe knowledge of the inventors, the existence of a general system, whichis termed “cross-β structure pathway,” to remove unwanted biomoleculesis herein disclosed for the first time. The results show that thismechanism is fundamental to nature and controls many physiologicalprocesses to protect organisms from induced damage or from accumulatinguseless or denatured biomolecules. If deregulated, by whatever means,this system may cause severe problems. On the other hand, if properlyused, this system may be applicable for inducing cell death in specifictarget cells, like, for example, tumor cells.

TABLES TABLE I Percentage β-sheet, as calculated front CD spectraIncubation time β-sheet Sample^(‡) (weeks) (%)^(†) Aβ (16-22) 100Albumin-glyceraldehyde 2 0 Albumin control 2 0 Albumin-g6p 2 0Albumin-g6p 4 7 Albumin control 23 0 Albumin-g6p 23 19^(‡)Two-weeks incubated albumin was from a different batch than four-and 23-weeks incubated albumin.^(†)Percentage of amino acid residues in β-sheets are given.

TABLE II Correlation between Hb_(A1c) concentrations and Hb fibrilformation in vitro Healthy controls Diabetes mellitus patients sample[Hb_(A1c)] (%)^(‡) Fibers^(†) sample [Hb_(A1c)] (%)^(‡) Fibers^(†) 1 5.6− 1 5.5 − 2 5.9 − 2 5.8 − 3 6.2 − 3 5.8 − 4 10.7 − 5 11.3 − 6 11.6 − 712.4 + 8 12.5 − 9 12.5 − 10 12.6 + 11 12.7 − 12 12.8 − 13 13.3 + 1413.7 + 15 14.8 + 16 15.3 +^(†)The Hb_(A1c) concentration is given as a percentage of the totalamount of Hb present in erythrocytes of diabetes mellitus patients andof healthy controls. The s.d. is 2.3% of the values given.^(‡)Presence of fibers as determined with TEM.

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1-35. (canceled)
 36. A method for detecting a plaque involved in aconformational disease, the method comprising: contacting a sample withan antibody capable of binding a cross-β structure epitope; anddetecting binding of the antibody to the cross-β structure epitope. 37.A method for detecting a plaque involved in a conformational disease,the method comprising: contacting a sample with a cross-β structurebinding domain; and detecting binding of the cross-β structure bindingdomain to the cross-β structure.
 38. The method according to claim 36,wherein said conformational disease is Alzheimers or diabetes. 39-47.(canceled)
 48. A method for detecting cross-β structures in a sample,the method comprising: contacting the sample with a compound capable ofbinding cross-β structures; allowing the cross-β structures to bind tothe compound; and detecting a complex formed through binding of thecompound to the cross-β structures.
 49. The method according to claim48, wherein the sample is of a body fluid origin.
 50. The methodaccording to claim 49, wherein the body fluid is selected from the groupconsisting of blood, serum, liquor, and combinations of any thereof. 51.The method according to claim 48, wherein the compound capable ofbinding cross-β structures is: an antibody, a fragment, or a derivativethereof against cross-β structures; a tPA finger domain or a functionalequivalent thereof; or a multiligand receptor for cross-β structures.52. The method according to claim 48, wherein the compound capable ofbinding cross-β structures is provided on a solid phase.
 53. Adiagnostic device for carrying out the method according to claim 48, thediagnostic device comprising: a sample container for holding a sample;means for contacting the sample with a cross-β structure bindingcompound; a cross-β structure binding compound; and means for detectingbound cross-β structures.
 54. The diagnostic device of claim 53, furthercomprising means for separating unbound cross-β structures from boundcross-β structures.
 55. The diagnostic device of claim 53, wherein saidcross-β compound is provided on a solid phase. 56-57. (canceled)
 58. Themethod according to claim 37, wherein said conformational disease isAlzheimers or diabetes.
 59. The method according to claim 36, furthercomprising: immobilizing the antibody capable of binding the cross-βstructure epitope on a substrate.
 60. The method according to claim 37,wherein the cross-β structure binding domain is: an antibody, afragment, or a derivative thereof against cross-β structures; a tPAfinger domain or a functional equivalent thereof; or a multiligandreceptor for cross-β structures.
 61. The method according to claim 36,wherein the sample is of a body fluid origin.
 62. The method accordingto claim 61, wherein the body fluid is selected from the groupconsisting of blood, serum, liquor, and combinations of any thereof. 63.The method according to claim 37, wherein the sample is of a body fluidorigin.
 64. The method according to claim 63, wherein the body fluid isselected from the group consisting of blood, serum, liquor, andcombinations of any thereof.
 65. The method according to claim 37,further comprising: immobilizing the cross-β structure binding domain ona substrate.